Immunogenic Mycoplasma hyopneumoniae polypeptides

ABSTRACT

Mycoplasma hyopneumoniae  polypeptides and nucleic acids, as well as nucleic acid expression vectors and host cells containing nucleic acid vectors are provided. In addition, compositions containing  M. hyopneumoniae  polypeptides and nucleic acids are provided for use in methods of treating swine to prevent enzootic pneumonia. Furthermore, the invention provides diagnostic tests for the detecting of  M. hyopneumoniae  infection in swine herds.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. §119(e) of U.S.application Ser. No. 60/392,632, filed Jun. 28, 2002.

BACKGROUND

[0002] 1. Technical Field

[0003] The invention relates to methods and materials involved inprotecting an animal against enzootic pneumonia.

[0004] 2. Background Information

[0005] Enzootic pneumonia in swine, also called mycoplasmal pneumonia,is caused by Mycoplasma hyopneumoniae. The disease is chronic andnon-fatal, affecting pigs of all ages. Although infected pigs show onlymild symptoms of coughs and fever, the disease has significant economicimpact due to reduced feed efficiency and reduced weight gain. Enzooticpneumonia is transmitted by airborne organisms expelled from the lungsof infected pigs. The primary infection by M. hyopneumoniae may befollowed by a secondary infection of other Mycoplasma species, e.g.,Mycoplasma hyorhinis and Mycoplasma flocculare, as well as otherbacterial pathogens.

[0006]M. hyopneumoniae infects the respiratory tracts of pigs,colonizing the tracheae, bronchi, and bronchioles. The pathogen producesa ciliostatic factor that causes the cilia lining the respiratorypassages to stop beating. Eventually, the cilia degenerate, leaving pigsprone to infection by secondary pathogens. Characteristic lesions ofpurple to gray areas of consolidation are observed in infected pigs.Surveys of slaughtered pigs revealed lesions in 30% to 80%. Results from37 herds in 13 states indicated that 99% of the herds had pigs withpneumonia lesions typical of enzootic pneumonia. Therefore, there is aneed for effective preventative and treatment measures.

[0007] Mycoplasmas vary their surface structure by a complex series ofgenetic events to present a structural mosaic to the host immune system.Phase switching of surface molecules occurs through a variety ofmechanisms such as changes in the number of repetitive units during DNAreplication, genomic inversions, transposition events, and/or geneconversion. See, for example, Zhang and Wise, 1997, Mol. Microbiol.,25:859-69; Theiss and Wise, 1997, J. Bacteriol., 179:4013-22; Sachse etal., 2000, Infect. Immun., 68:680-7; Dybvig and Uy, 1994, Mol.Microbiol., 12:547-60; and Lysnyansky et al., 1996, J. Bacteriol.,178:5395-5401. All of the identified phase variable and phase switchinggenes in mycoplasmas that code for surface proteins are lipoproteins.

SUMMARY

[0008] The invention provides materials and methods for protecting ananimal from enzootic pneumonia. The invention is based on the discoveryof Mycoplasma hyopneumoniae nucleic acids that encode cell surfacepolypeptides that can be used for inducing a protective immune responsein an animal susceptible to pneumonia. More specifically, the inventionprovides purified immunogenic polypeptides of these polypeptides forused to as antigens for illiciting an immune response in an animal, e.g.a pig. In addition, the invention also provides isolated nucleic acidsencoding these immunogenic polypeptides for use in generating an immuneresponse in an animal. Purified polypeptides and isolated nucleic acidsof the invention can be combined with pharmaceutically acceptablecarriers for introducing into an animal. The invention also providesmaterials and methods for determining whether an animal has an antibodyreactive to the polypeptides of the invention.

[0009] In one aspect, the invention provides a purified immunogenicpolypeptide, the amino acid sequence of which comprises at least eightconsecutive residues of a sequence selected from the group consisting ofSEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, and 20. Specifically, theinvention provides an immunogenic polypeptide of the invention, theamino acid sequence of which comprises at least eight consecutiveresidues of SEQ ID NO: 2; an immunogenic polypeptide of the invention,the amino acid sequence of which comprises at least eight consecutiveresidues of SEQ ID NO:4; an immunogenic polypeptide of the invention,the amino acid sequence of which comprises at least eight consecutiveresidues of SEQ ID NO:6; an immunogenic polypeptide of the invention,the amino acid sequence of which comprises at least eight consecutiveresidues of SEQ ID NO:8; an immunogenic polypeptide of the invention,the amino acid sequence of which comprises at least eight consecutiveresidues of SEQ ID NO:10; an immunogenic polypeptide of the invention,the amino acid sequence of which comprises at least eight consecutiveresidues of SEQ ID NO:12; an immunogenic polypeptide of the invention,the amino acid sequence of which comprises at least eight consecutiveresidues of SEQ ID NO:14; an immunogenic polypeptide of the invention,the amino acid sequence of which comprises at least eight consecutiveresidues of SEQ ID NO:16; an immunogenic polypeptide of the invention,the amino acid sequence of which comprises at least eight consecutiveresidues of SEQ ID NO:18; an immunogenic polypeptide of the invention,the amino acid sequence of which comprises at least eight consecutiveresidues of SEQ ID NO: 20.

[0010] In another aspect, the invention provides mutants of theabove-described immunogenic polypeptides, wherein such mutantpolypeptides retain immunogenicity.

[0011] Generally, immunogenic polypeptides and immunogenic mutantpolypeptides of the invention include at least 8 consecutive residues(e.g., at least 10, 12, 15, 20, or 25) of SEQ ID NOs:2, 4, 6, 8, 10, 12,14, 16, 18, or 20.

[0012] In another aspect, the invention provides a composition thatincludes one or more of the above-described immunogenic polypeptides orimmunogenic mutant polypeptides.

[0013] In one aspect, the invention provides a method of eliciting animmune response in an animal. Such a method includes introducing acomposition comprising the above-described immunogenic polypeptides orimmunogenic mutant polypeptides into the animal. Such a composition canbe administered orally, intranasally, intraperitoneally,intramuscularly, subcutaneously, or intravenously. A representativeanimal into which the compositions of the invention can be introduced isa swine.

[0014] In another aspect, the invention provides an isolated nucleicacid comprising a nucleotide sequence that encodes an immunogenicpolypeptide, the amino acid sequence of which comprises at least eightconsecutive residues of a sequence such as SEQ ID NOs: 2, 4, 6, 8, 10,12, 14, 16, 18, or 20. The invention also features mutants of nucleicacids that encode an immunogenic polypeptide. Representative nucleicacids encoding such immunogenic polypeptides have a nucleotide sequenceas shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19,respectively.

[0015] Specifically, the invention provides a nucleic acid having anucleotide sequence encoding an immunogenic polypeptide, the amino acidsequence of which comprises at least eight consecutive residues of SEQID NO:2. A representative nucleic acid encoding such a polypeptide hasthe nucleotide sequence of SEQ ID NO:1.

[0016] Specifically, the invention provides a nucleic acid having anucleotide sequence encoding an immunogenic polypeptide, the amino acidsequence of which comprises at least eight consecutive residues of SEQID NO:4. A representative nucleic acid encoding such a polypeptide hasthe nucleotide sequence of SEQ ID NO:3.

[0017] Specifically, the invention provides a nucleic acid having anucleotide sequence encoding an immunogenic polypeptide, the amino acidsequence of which comprises at least eight consecutive residues of SEQID NO:6. A representative nucleic acid encoding such a polypeptide hasthe nucleotide sequence of SEQ ID NO:5.

[0018] Specifically, the invention provides a nucleic acid having anucleotide sequence encoding an immunogenic polypeptide, the amino acidsequence of which comprises at least eight consecutive residues of SEQID NO:8. A representative nucleic acid encoding such a polypeptide hasthe nucleotide sequence of SEQ ID NO:7.

[0019] Specifically, the invention provides a nucleic acid having anucleotide sequence encoding an immunogenic polypeptide, the amino acidsequence of which comprises at least eight consecutive residues of SEQID NO:10. A representative nucleic acid encoding such a polypeptide hasthe nucleotide sequence of SEQ ID NO:9.

[0020] Specifically, the invention provides a nucleic acid having anucleotide sequence encoding an immunogenic polypeptide, the amino acidsequence of which comprises at least eight consecutive residues of SEQID NO:12. A representative nucleic acid encoding such a polypeptide hasthe nucleotide sequence of SEQ ID NO:11.

[0021] Specifically, the invention provides a nucleic acid having anucleotide sequence encoding an immunogenic polypeptide, the amino acidsequence of which comprises at least eight consecutive residues of SEQID NO:14. A representative nucleic acid encoding such a polypeptide hasthe nucleotide sequence of SEQ ID NO:13.

[0022] Specifically, the invention provides a nucleic acid having anucleotide sequence encoding an immunogenic polypeptide, the amino acidsequence of which comprises at least eight consecutive residues of SEQID NO:16. A representative nucleic acid encoding such a polypeptide hasthe nucleotide sequence of SEQ ID NO:15.

[0023] Specifically, the invention provides a nucleic acid having anucleotide sequence encoding an immunogenic polypeptide, the amino acidsequence of which comprises at least eight consecutive residues of SEQID NO:18. A representative nucleic acid encoding such a polypeptide hasthe nucleotide sequence of SEQ ID NO:17.

[0024] Specifically, the invention provides a nucleic acid having anucleotide sequence encoding an immunogenic polypeptide, the amino acidsequence of which comprises at least eight consecutive residues of SEQID NO:20. A representative nucleic acid encoding such a polypeptide hasthe nucleotide sequence of SEQ ID NO:19.

[0025] The invention also provides a vector containing a nucleic acid ofthe invention. A vector can further include an expression controlsequence operably linked to the nucleic acid. The invention additionallyprovides host cells comprising such vectors. The invention furtherprovides a composition that includes such vectors and a pharmaceuticallyacceptable carrier.

[0026] In yet another aspect, the invention provides a method ofeliciting an immune response in an animal. Such a method includesintroducing a composition of the invention into the animal. Suchcompositions can be administered orally, intranasally,intraperitoneally, intramuscularly, subcutaneously, or intravenously.Generally, the animal is a swine.

[0027] In still yet another aspect, the invention provides a method ofdetermining whether or not an animal has an antibody reactive to animmunogenic polypeptide of the invention, the method comprising:providing a test sample from the animal; contacting the test sample withthe immunogenic polypeptide under conditions permissible for specificbinding of the immunogenic polypeptide with the antibody; and detectingthe presence or absence of the specific binding. Typically, the presenceof specific binding indicates that the animal has the antibody, and theabsence of specific binding indicates that the animal does not have theantibody.

[0028] Generally, an appropriate test sample is a biological fluid suchas blood, nasal fluid, throat fluid, or lung fluid. In some embodiments,the immunogenic polypeptide is attached to a solid support such as amicrotiter plate, or polystyrene beads. In some embodiments, theimmunogenic polypeptide is labeled. By way of example, the detectingstep can be by radioimmunoassay (RIA), enzyme immunoassay (EIA), orenzyme-linked immunosorbent assay (ELISA).

[0029] In another aspect, the invention provides a diagnostic kit fordetecting the presence of an antibody in a test sample, wherein such anantibody is reactive to an immunogenic polypeptide of the invention.Such a kit can include one or more of the immunogenic polypeptides ofthe invention.

[0030] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

[0031] Other features and advantages of the invention will be apparentfrom the following detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

[0032]FIG. 1 is the nucleic acid sequence encoding C2-mhp210 (SEQ IDNO:1), a P102 paralog from M. hyopneumoniae strain 232.

[0033]FIG. 2 is the polypeptide sequence of C2-MHP210 (SEQ ID NO:2) fromM. hyopneumoniae strain 232.

[0034]FIG. 3 is the nucleic acid sequence encoding C2-mhp211 (SEQ IDNO:3) from M. hyopneumoniae strain 232.

[0035]FIG. 4 is the polypeptide sequence of C2-MHP211 (SEQ ID NO:4) fromM. hyopneumoniae strain 232.

[0036]FIG. 5 is the nucleic acid sequence encoding C27-mhp348 (SEQ IDNO:5), a P102 paralog from M. hyopneumoniae strain 232.

[0037]FIG. 6 is the polypeptide sequence of C27-MHP348 (SEQ ID NO:6)from M. hyopneumoniae strain 232.

[0038]FIG. 7 is the nucleic acid sequence encoding C28-mhp545 (SEQ IDNO:7) from M. hyopneumoniae strain 232.

[0039]FIG. 8 is the polypeptide sequence of C28-MHP545 (SEQ ID NO:8)from M. hyopneumoniae strain 232.

[0040]FIG. 9 is the nucleic acid sequence encoding C28-mhp662 (SEQ IDNO:9) from M. hyopneumoniae strain 232.

[0041]FIG. 10 is the polypeptide sequence of C28-MHP662 (SEQ ID NO:10)from M. hyopneumoniae strain 232.

[0042]FIG. 11 is the nucleic acid sequence encoding C28-mhp663 (SEQ IDNO:11), a P102 paralog from M. hyopneumoniae strain 232.

[0043]FIG. 12 is the polypeptide sequence of C28-MHP663 (SEQ ID NO:12)from M. hyopneumoniae strain 232.

[0044]FIG. 13 is the nucleic acid sequence encoding C2-mhp036 (SEQ IDNO: 13), a P102 paralog from M. hyopneumoniae strain 232.

[0045]FIG. 14 is the polypeptide sequence of C2-MPH036 (SEQ ID NO:14)from M. hyopneumoniae strain 232.

[0046]FIG. 15 is the nucleic acid sequence encoding C2-mhp033 (SEQ IDNO: 15), a partial paralog of P102 from M. hyopneumoniae strain 232.

[0047]FIG. 16 is the polypeptide sequence of C2-MHP033 (SEQ ID NO:16)from M. hyopneumoniae strain 232.

[0048]FIG. 17 is the nucleic acid sequence encoding C2-mhp034 (SEQ IDNO: 17), a partial paralog of P102 from M. hyopneumoniae strain 232.

[0049]FIG. 18 is the polypeptide sequence of C2-MHP034 (SEQ ID NO:18)from M. hyopneumoniae strain 232.

[0050]FIG. 19 is the nucleic acid sequence encoding C28-mhp545 (SEQ IDNO:19) from M. hyopneumoniae strain J.

[0051]FIG. 20 is the polypeptide sequence of C28-MHP545 (SEQ ID NO:20)from M. hyopneumoniae strain J.

[0052]FIG. 21 is the structure of P102 paralogs and their organizationin the chromosome.

[0053]FIG. 22 shows a map and hydrophilicity plot of P216. The upperpanel depicts a schematic diagram of the P216 protein sequence.Asterisks indicate locations of peptides used to clone the gene (left,amino acids 94-105) and used to make antisera specific for P130 (right,amino acids 1654-1668). The arrow indicates the position of the majorcleavage event. The gray box indicates the position of the 30-kDafragment cloned and expressed (amino acids 1043-1226). The invertedfilled triangles are locations of tryptophan residues encoded by TGAcodons. The hatched boxes are the location of the coiled coil domains.The white box indicates the location of the BNBD (amino acids1012-1029). The black box represents the transmembrane domain (aminoacids 7-30). The lower panel represents the hydrophilicity plot.

DETAILED DESCRIPTION

[0054] The following abbreviations are used in this application: aa,amino acid(s); Ab, antibody(ies); bp, base pair(s); CHEF, clampedhomogenous electric field; H., Haemophilus; kb, kilobase(s) or 1000 bp;Kn, kanamycin; LB, Luria-Bertoni media; M., Mycoplasma; mAb, monoclonalAb; ORF, open reading frame; PCR, polymerase chain reaction; ^(R),resistant/resistance; Tn, transposon(s); ::, novel junction (fusion orinsertion). One letter and three letter code designations for aminoacids are given in Table 1. TABLE 1 Amino Acid Code Designations ThreeOne letter Letter Amino Acid code code Alanine Ala A Arginine Arg RAsparagine Asn N Aspartic Acid Asp D Cysteine Cys C Glutamic Acid Glu EGlutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile I LeucineLeu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro PSerine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine ValV

[0055]M. hyopneumoniae Polypeptides and Nucleic Acids

[0056] As used herein, the term “polypeptide” refers to a polymer ofthree or more amino acids covalently linked by amide bonds. Apolypeptide may or may not be post-translationally modified. As usedherein, the term “purified polypeptide” refers to a polypeptidepreparation that is substantially free of cellular material or othercontaminating polypeptides from the cell or tissue source from which thepolypeptide is derived, or substantially free of chemical precursors orother chemicals when chemically synthesized. For example, a polypeptidepreparation is substantially free of cellular material when thepolypeptide is separated from components of the cell from which thepolypeptide is obtained or recombinantly produced. Thus, a polypeptidepreparation that is substantially free of cellular material includes,for example, a preparation having less than about 30%, 20%, 10%, or 5%(dry weight) of heterologous polypeptides (also referred to herein as a“contaminating polypeptides”). When a polypeptide is recombinantlyproduced, the polypeptide is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,10%, 5% of the volume of the polypeptide preparation. When a polypeptideis produced by chemical synthesis, it is preferably substantially freeof chemical precursors or other chemicals, i.e., it is separated fromchemical precursors or other chemicals that are involved in thesynthesis of the polypeptide. Accordingly, such polypeptide preparationshave less than about 30%, 20%, 10%, 5% (by dry weight) of chemicalprecursors or compounds other than the polypeptide of interest.

[0057] As used herein, the term “mutant” refers to a polypeptide, or anucleic acid encoding a polypeptide, that has one or more conservativeamino acid variations or other minor modifications such that (1) thecorresponding polypeptide has substantially equivalent function whencompared to the wild type polypeptide or (2) an antibody raised againstthe polypeptide is immunoreactive with the wild-type polypeptide.

[0058] The term “conservative variation” denotes the replacement of anamino acid residue by another biologically similar residue, or thereplacement of a nucleotide in a nucleic acid sequence such that theencoded amino acid residue does not change or is another biologicallysimilar residue. Examples of conservative variations include thesubstitution of one hydrophobic residue such as isoleucine, valine,leucine or methionine for another hydrophobic residue, or thesubstitution of one polar residue for another polar residue, such as thesubstitution of arginine for lysine, glutamic for aspartic acid, orglutamine for asparagine, and the like. The term “conservativevariation” also includes the use of a substituted amino acid in place ofan unsubstituted parent amino acid provided that antibodies raised tothe substituted polypeptide also immunoreact with the unsubstitutedpolypeptide.

[0059] Any M. hyopneumoniae strain may be used as a starting material toproduce the polypeptides and nucleic acids of the present invention.Suitable strains of M. hyopneumoniae may be obtained from a variety ofsources, including depositories such as the American Type CultureCollection (ATCC) (Manassas, Va.) and the NRRL Culture Collection(Agricultural Research Service, U.S. Department of Agriculture, Peoria,Ill.). M. hyopneumoniae strains may also be obtained from lungsecretions or tissues from sick animals followed by inoculating suitableculture media.

[0060] An immunogenic polypeptide of the present invention can have anamino acid sequence shown in FIG. 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20.Alternatively, an immunogenic polypeptide of the present invention canbe a fragment of a polypeptide that has an amino acid sequence shown inFIG. 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20. An immunogenic polypeptideof the invention can be six or more, or preferably eight or more, aminoacids in length, but less than the full-length number of amino acids.For example, an immunogenic polypeptide can be 10, 12, 15, 20, 25, 30,or greater than 30 amino acids in length. A polypeptide of the presentinvention also can be a mutant of a polypeptide having an amino acidsequence shown in FIG. 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20. Mutationsat either the amino acid or nucleic acid level may be useful inimproving the yield of the polypeptides, their immunogenicity orantigenicity, or their compatibility with various expression systems,adjuvants and modes of administration. Synthetic or recombinantfragments of wild type or mutated polypeptides are characterized by oneor more of the antigenic sites of native M. hyopneumoniae polypeptides,the sequences of which are illustrated in FIGS. 2, 4, 6, 8, 10, 12, 14,16, 18, and 20.

[0061] The polypeptides of the present invention may be obtained from M.hyopneumoniae cells or may be produced in host cells transformed bynucleic acids that encode these polypeptides. Recombinant polypeptidesproduced from transformed host cells may include residues that are notrelated to M. hyopneumoniae. For example, a recombinant polypeptide maybe a fusion polypeptide containing an amino acid portion derived from anexpression vector, or other source, in addition to the portion derivedfrom M. hyopneumoniae. A recombinant polypeptide may also include astarting methionine. Recombinant polypeptides of the invention displaythe antigenicity of native M. hyopneumoniae polypeptides the sequencesof which are illustrated in FIGS. 2, 4, 6, 8, 10, 12, 14, 16, 18, and20.

[0062] Nucleic acid sequences encoding full-length polypeptides of thepresent invention are shown in FIGS. 1, 3, 5, 7, 9, 11, 13, 15, 17, and19. The present invention encompasses nucleic acid sequences, as well asfragments or mutants of these, that encode immunogenic polypeptides,i.e., capable of eliciting antibodies or other immune responses (e.g.,T-cell responses of the immune system) that recognize epitopes of thepolypeptides having sequences illustrated in FIGS. 2, 4, 6, 8, 10, 12,14, 16, 18, and 20. Hence, nucleic acid sequences of the presentinvention may encode polypeptides that are full-length polypeptides,polypeptide fragments, and mutant or fusion polypeptides.

[0063] The term “nucleic acid” as used herein encompasses RNA and DNA,including cDNA, genomic DNA, and synthetic (e.g., chemicallysynthesized) DNA. The nucleic acid can be double-stranded orsingle-stranded. Where single-stranded, the nucleic acid can be thesense strand or the antisense strand. In addition, nucleic acid can becircular or linear.

[0064] The term “isolated” as used herein with reference to nucleic acidrefers to a naturally-occurring nucleic acid that is not immediatelycontiguous with both of the sequences with which it is immediatelycontiguous (one on the 5′ end and one on the 3′ end) in thenaturally-occurring genome of the organism from which it is derived. Forexample, an isolated nucleic acid can be, without limitation, arecombinant DNA molecule of any length, provided one of the nucleic acidsequences normally found immediately flanking that recombinant DNAmolecule in a naturally-occurring genome is removed or absent. Thus, anisolated nucleic acid includes, without limitation, a recombinant DNAthat exists as a separate molecule (e.g., a cDNA or a genomic DNAfragment produced by PCR or restriction endonuclease treatment)independent of other sequences as well as recombinant DNA that isincorporated into a vector, an autonomously replicating plasmid, a virus(e.g., a retrovirus, adenovirus, or herpes virus), or into the genomicDNA of a prokaryote or eukaryote. In addition, an isolated nucleic acidcan include a recombinant DNA molecule that is part of a hybrid orfusion nucleic acid sequence.

[0065] The term “isolated” as used herein with reference to nucleic acidalso includes any non-naturally-occurring nucleic acid sincenon-naturally-occurring nucleic acid sequences are not found in natureand do not have immediately contiguous sequences in a naturallyoccurring genome. For example, non-naturally-occurring nucleic acid suchas an engineered nucleic acid is considered to be isolated nucleic acid.Engineered nucleic acid can be made using common molecular cloning orchemical nucleic acid synthesis techniques. Isolatednon-naturally-occurring nucleic acid can be independent of othersequences, or incorporated into a vector, an autonomously replicatingplasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), orthe genomic DNA of a prokaryote or eukaryote. In addition, anon-naturally-occurring nucleic acid can include a nucleic acid moleculethat is part of a hybrid or fusion nucleic acid sequence.

[0066] It will be apparent to those of skill in the art that a nucleicacid existing among hundreds to millions of other nucleic acid moleculeswithin, for example, cDNA or genomic libraries, or gel slices containinga genomic DNA restriction digest is not to be considered an isolatednucleic acid.

[0067] The term “exogenous” as used herein with reference to nucleicacid and a particular cell refers to any nucleic acid that does notoriginate from that particular cell as found in nature. Thus,non-naturally-occurring nucleic acid is considered to be exogenous to acell once introduced into the cell. It is important to note thatnon-naturally-occurring nucleic acid can contain nucleic acid sequencesor fragments of nucleic acid sequences that are found in nature providedthe nucleic acid as a whole does not exist in nature. For example, anucleic acid molecule containing a genomic DNA sequence within anexpression vector is non-naturally-occurring nucleic acid, and thus isexogenous to a cell once introduced into the cell, since that nucleicacid molecule as a whole (genomic DNA plus vector DNA) does not exist innature. Thus, any vector, autonomously replicating plasmid, or virus(e.g., retrovirus, adenovirus, or herpes virus) that as a whole does notexist in nature is considered to be non-naturally-occurring nucleicacid. It follows that genomic DNA fragments produced by PCR orrestriction endonuclease treatment as well as cDNAs are considered to benon-naturally-occurring nucleic acid since they exist as separatemolecules not found in nature. It also follows that any nucleic acidcontaining a promoter sequence and polypeptide-encoding sequence (e.g.,cDNA or genomic DNA) in an arrangement not found in nature isnon-naturally-occurring nucleic acid.

[0068] Nucleic acid that is naturally occurring can be exogenous to aparticular cell. For example, an entire chromosome isolated from a cellof person X is an exogenous nucleic acid with respect to a cell ofperson Y once that chromosome is introduced into Y's cell.

[0069] Recombinant nucleic acid molecules that are useful in preparingthe aforementioned polypeptides are also provided. Preferred recombinantnucleic acid molecules include, without limitation, (1) those havingnucleic acid sequences illustrated in FIGS. 1, 3, 5, 7, 9, 11, 13, 15,17, and 19; (2) cloning or expression vectors containing sequencesencoding recombinant polypeptides of the present invention; (3) nucleicacid sequences that hybridize to those sequences that encode M.hyopneumoniae polypeptides of the invention; (4) degenerate nucleic acidsequences that encode polypeptides of the invention.

[0070] Nucleic acids of the invention may be inserted into any of a widevariety of expression vectors by a variety of procedures, generallythrough use of an appropriate restriction endonuclease site. Suitablevectors include, for example, vectors consisting of segments ofchromosomal, non-chromosomal and synthetic nucleic acid sequences, suchas various known derivatives of SV40; known bacterial plasmids, e.g.,plasmids from E. coli including col E1, pCR1, pBR322, pMB9 and theirderivatives; wider host range plasmids, e.g., RP4; phage DNAs, e.g., thenumerous derivatives of phage λ, e.g., NM 989, and other DNA phages suchas M13 or filamentous single stranded DNA phages; yeast plasmids such asthe 2μ plasmid or derivatives thereof; viral DNA such as baculovirus,vaccinia, adenovirus, fowl pox virus, or pseudorabies; and vectorsderived from combinations of plasmids and phage DNAs, such as plasmidswhich have been modified to employ phage DNA or other expression controlsequences.

[0071] Within each specific cloning or expression vector, various sitesmay be selected for insertion of the nucleic acids of this invention.These sites are usually designated by the restriction endonuclease thatcuts them, and there are various known methods for inserting nucleicacids into these sites to form recombinant molecules. These methodsinclude, for example, dG-dC or dA-dT tailing, direct ligation, syntheticlinkers, exonuclease and polymerase-linked repair reactions followed byligation, or extension of the nucleic acid strand with DNA polymeraseand an appropriate single-stranded template followed by ligation. It isto be understood that a cloning or expression vector useful in thisinvention need not have a restriction endonuclease site for insertion ofthe chosen nucleic acid fragment, and that insertion may occur byalternative means.

[0072] For expression of the nucleic acids of this invention, thesenucleic acid sequences are operatively linked to one or more expressioncontrol sequences in the expression vector. Such operative linking,which may be effected before or after the chosen nucleic acid isinserted into a cloning vehicle, enables the expression controlsequences to control and promote the expression of the inserted nucleicacid.

[0073] Any of a wide variety of expression control sequences—sequencesthat control the expression of a nucleic acid when operatively linked toit—may be used in these vectors to express the nucleic acid sequences ofthis invention. Such useful expression control sequences include, forexample, the early and late promoters of SV40, the lac or trp systems,the TAC or TRC system, the major operator and promoter regions of λ, thecontrol regions of fd coat protein, the promoter for 3-phosphoglyceratekinase or other glycolytic enzymes, the promoters of acid phosphatase,e.g., Pho5, the promoters of the yeast α-mating factors, and othersequences known to control the expression of genes in prokaryotic oreukaryotic cells or their viruses, and various combinations thereof. Theexpression vector also includes a non-coding sequence for aribosome-binding site for translation initiation and a transcriptionterminator. The vector may also include appropriate sequences foramplifying expression. In mammalian cells, it is additionally possibleto amplify the expression units by linking the gene to that coding fordehydrofolate reductase and applying a selection to host Chinese hamsterovary cells.

[0074] The vector or expression vehicle, and in particular, the siteschosen therein for insertion of the selected nucleic acid fragment, andthe expression control sequence employed in this invention aredetermined by a variety of factors, e.g., number of sites susceptible toa particular restriction enzyme, size of the polypeptide to beexpressed, expression characteristics such as the location of start andstop codons relative to the vector sequences, and other factorsrecognized by those of skill in the art. The choice of a vector,expression control sequence, and/or insertion site are determined by abalance of these factors, as not all selections are equally effectivefor a given case.

[0075] The recombinant nucleic acid molecule containing the desiredcoding sequence operatively linked to an expression control sequence maythen be employed to transform a wide variety of appropriate hosts so asto permit such hosts (transformants) to express the coding sequence, orfragment thereof, and to produce the polypeptide, or portion thereof,for which the hybrid nucleic acid encodes. The recombinant nucleic acidmolecule may also be employed to transform a host so as to permit thathost on replication to produced additional recombinant nucleic acidmolecules as a source of M. hyopneumoniae coding sequences and fragmentsthereof.

[0076] A wide variety of hosts are also useful in producing polypeptidesand nucleic acids of this invention. These hosts include, for example,bacteria such as E. coli, Bacillus and Streptomyces, fungi such asyeasts, and animal or plant cells in tissue culture. The selection of anappropriate host for these uses is controlled by a number of factors.These include, for example, compatibility with the chosen vector,toxicity of the co-products, ease of recovery of the desiredpolypeptide, expression characteristics, biosafety and costs. Noabsolute choice of host may be made for a particular recombinant nucleicacid molecule or polypeptide from any of these factors alone. Instead, abalance of these factors is applied with the realization that not allhosts may be equally effective for expression of a particularrecombinant nucleic acid molecule.

[0077] It is also understood that the nucleic acid sequences that areinserted at the selected site of a cloning or expression vector mayinclude nucleotides that are not part of the actual coding sequence forthe desired polypeptide or may include only a fragment of the entirecoding sequence for that polypeptide. It is only required that whateverDNA sequence is employed, the transformed host produces a polypeptidehaving the antigenicity of native M. hyopneumoniae polypeptides.

[0078] For example, in an expression vector of this invention, a nucleicacid of this invention may be fused in the same reading frame to aportion of a nucleic acid sequence coding for at least one eukaryotic orprokaryotic carrier polypeptide or a nucleic acid sequence coding for atleast one eukaryotic or prokaryotic signal sequence, or combinationsthereof. Such constructions may aid in expression of the desired nucleicacid sequence or improve purification, permit secretion, and preferablymaturation of the desired polypeptide from the host cell. The nucleicacid sequence may alternatively include an ATG start codon, alone, ortogether with other codons, fused directly to the sequence encoding thefirst amino acid of a desired polypeptide. Such constructions enable theproduction of, for example, a methionyl or other peptidyl polypeptidethat is part of this invention. This N-terminal methionine or peptidemay then be cleaved intracellularly or extracellularly by a variety ofknown processes or the polypeptide used together with the methionine orother fusion attached to it in the compositions and methods of thisinvention.

[0079] The appropriate nucleic acid sequence present in the vector whenintroduced into a host may express part or only a portion of thepolypeptide that is encoded, it being sufficient that the expressedpolypeptide be capable of eliciting an antibody or other immune responsethat recognizes an epitope of the amino acid sequence depicted in FIG.2, 4, 6, 8, 10, 12, 14, 16, 18, or 20. For example, in employing E. colias a host organism, the UGA codon is a stop codon so that the expressedpolypeptide may only be a fragment of the polypeptide encoded by thevector, and therefore, it is generally preferred that all of the UGAcodons in the appropriate nucleic acid sequence be converted intonon-stop codons. Alternatively, an additional nucleic acid sequence thatencodes a t-RNA that translates the UGA codon into a tryptophan residuecan be introduced into the host.

[0080] The polypeptide expressed by the host transformed by the vectormay be harvested by methods known to those skilled in the art, and usedfor protection of a non-human animal such as swine, cattle, etc. againstenzootic pneumonia caused by M. hyopneumoniae. The polypeptide is usedin an amount effective to provide protection against enzootic pneumoniacaused by M. hyopneumoniae and may be used in combination with asuitable physiologically acceptable carrier as described below.

[0081] Detecting M. hyopneumoniae

[0082] The polypeptides of the present invention may also be used asantigens for diagnostic purposes to determine whether a biological testsample contains M. hyopneumoniae antigens or antibodies to theseantigens. Such assays for M. hyopneumoniae infection in an animaltypically involve incubating an antibody-containing biological samplefrom an animal suspected of having such a condition in the presence of adetectably labeled polypeptide of the present invention, and detectingbinding. The immunogenic polypeptide is generally present in an amountthat is sufficient to produce a detectable level of binding withantibody present in the antibody-containing sample.

[0083] Thus, in this aspect of the invention, the polypeptide may beattached to a solid phase support, e.g., a microtiter plate, which iscapable of immobilizing cells, cell particles or soluble polypeptides.The support may then be washed with suitable buffers followed bytreatment with the sample from the animal. The solid phase support maythen be washed with the buffer a second time to remove unbound antibody.Labeled polypeptide is added and the support is washed a third time toremove unbound labeled polypeptide. The amount of bound label on saidsolid support may then be detected by conventional means.

[0084] By “solid phase support” is intended any support capable ofbinding antigen or antibodies. Well-known supports, or carriers, includeglass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses (especially nitrocellulose),polyacrylamides, agarose, and magnetite. The nature of the carrier canbe either soluble to some extent or insoluble for the purposes of thepresent invention. The support material may have virtually any possiblestructural configuration so long as the coupled molecule is capable ofbinding to an antigen or antibody. Thus, the support configuration maybe spherical, as in a bead, or cylindrical, as in the inside surface ofa test tube, or the external surface of a rod. Alternatively, thesurface may be flat such as for example, a sheet or test strip.Preferred supports include polystyrene beads.

[0085]M. hyopneumoniae specific antibody can be detectably labeled bylinking the same to an enzyme and using it in an enzyme immunoassay(EIA), or enzyme-linked immunosorbent assay (ELISA). This enzyme, inturn, when later exposed to its substrate, will react with the substratein such a manner as to produce a chemical moiety that can be detected,for example, by spectrophotometric, fluorometric or by visual means.Enzymes that can be used to detectably label the M. hyopneumoniaespecific antibody include, but are not limited to, horseradishperoxidase, malate dehydrogenase, staphylococcal nuclease,delta-V-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate dehydrogenase, triose phosphate isomerase,alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase,ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,glucoa/nylase and acetylcholinesterase.

[0086] Detection may be accomplished using any of a variety ofimmunoassays. For example, by radioactively labeling the recombinantprotein, it is possible to detect antibody binding through aradioimmunoassay (RIA). The radioactive isotope can be detected by suchmeans as the use of a gamma counter or a scintillation counter or byautoradiography. Isotopes which are particularly useful for the purposeof the present invention include ³H, ¹²⁵I, ¹³¹I, ³⁵S, and ¹⁴C,preferably ¹²⁵I.

[0087] It is also possible to label the recombinant polypeptide with afluorescent compound. When the fluorescently labeled polypeptide isexposed to light of the proper wavelength, its presence can then bedetected due to fluorescence. Among the most commonly used fluorescentlabeling compounds are fluorescein isothiocyanate, rhodamine,phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde andfluorescamine. The polypeptide can also be detectably labeled usingfluorescence emitting metals such as ¹⁵²Eu, or others of the lanthanideseries. These metals can be attached to the protein using such metalchelating groups as diethylenetriaminepentaacetic acid (DTPA) orethylenediamine-tetraacetic acid (EDTA).

[0088] The polypeptide also can be detectably labeled by coupling it toa chemiluminescent or bioluminescent compound. The presence of thechemiluminescent-tagged polypeptide is then determined by detecting thepresence of luminescence that arises during the course of a chemicalreaction. Bioluminescence is a type of chemiluminescence found inbiological systems in which a catalytic protein increases the efficiencyof the chemiluminescent reaction. Examples of particularly usefulchemiluminescent labeling compounds are luminol, isoluminol, theromaticacridinium ester, imidazole, acridinium salt and oxalate ester.Important bioluminescent compounds for purposes of labeling areluciferin, luciferase and aequorin.

[0089] Detection of the label may be accomplished by a scintillationcounter, for example, if the detectable label is a radioactive gammaemitter, or by a fluorometer, for example, if the label is a fluorescentmaterial. In the case of an enzyme label, the detection can beaccomplished by colorimetric methods that employ a substrate for theenzyme. Detection may also be accomplished by visual comparison of theextent of enzymatic reaction of a substrate in comparison with similarlyprepared standards.

[0090] The detection of foci of detectably labeled antibodies isindicative of a disease or dysfunctional state and may be used tomeasure M. hyopneumoniae in a sample. The absence of such antibodies orother immune response indicates that the animal has been neithervaccinated nor infected. For the purposes of the present invention, thebacterium that is detected by this assay may be present in a biologicalsample. Any sample containing it can be used, however, one of thebenefits of the present diagnostic invention is that invasive tissueremoval may be avoided. Therefore, preferably, the sample is abiological fluid such as, for example, blood, or nasal, throat or lungfluid, but the invention is not limited to assays using these samples.

[0091] In situ detection may be accomplished by removing a histologicalspecimen from an animal, and providing the combination of labeledantibodies of the present invention to such a specimen. The antibody (orfragment) is preferably provided by applying or by overlaying thelabeled antibody (or fragment) to a biological sample. Through the useof such a procedure, it is possible to determine not only the presenceof M. hyopneumoniae but also the distribution of it in the examinedtissue. Using the present invention, those of ordinary skill willreadily perceive that any of a wide variety of histological methods(such as staining procedures) can be modified in order to achieve suchin situ detection.

[0092] Alternatively, a sample (e.g., a fluid or tissue sample) may betested for the presence of a coding sequence for a M. hyopneumoniaepolypeptide of the invention by reaction with a recombinant or syntheticnucleic acid sequence contained within the sequence shown in FIGS. 1, 3,5, 7, 9, 11, 13, 15, 17, 19, or any RNA sequence equivalent to thisnucleic acid sequence. The absence of the coding sequence indicates thatthe animal has been neither vaccinated nor infected. This test involvesmethods of synthesis, amplification, or hybridization of nucleic acidsequences that are known to those skilled in the art. See, for example,Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2^(nd)Ed, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; PCR, APractical Approach, Vols 1 & 2, McPherson et al. (eds.), OxfordUniversity Press, 1992 and 1995; and PCR Strategies, Innis (ed.),Academic Press, 1995, herein incorporated by reference.

[0093] Compositions

[0094] The present invention also contemplates a composition (e.g., avaccine) comprising the recombinant polypeptides of the presentinvention, or nucleic acid sequences encoding these polypeptides, forimmunizing or protecting non-human animals, preferably swine, against M.hyopneumoniae infections, particularly enzootic pneumonia. The terms“protecting” or “protection” when used with respect to the compositionfor enzootic pneumonia described herein means that the compositionprevents enzootic pneumonia caused by M. hyopneumoniae and/or reducesthe severity of the disease. When a composition elicits an immunologicalresponse in an animal, the animal is considered seropositive, i.e., theanimal produces a detectable amount of antibodies against a polypeptideof the invention. Methods for detecting an immunological response in ananimal are well known.

[0095] Compositions generally include an immunologically effectivedosage of a polypeptide of the invention. An “immunologically effective”dosage is an amount that, when administered to an animal, elicits animmunological response in the animal but does not cause the animal todevelop severe clinical signs of an infection. An animal that hasreceived an immunologically effective dosage is an inoculated animal oran animal containing an inoculant of an immunologically effective amountof a polypeptide of the invention. Immunologically effective dosages canbe determined experimentally and may vary according to the type, size,age, and health of the animal vaccinated. The vaccination may include asingle inoculation or multiple inoculations. Other dosage schedules andamounts, including vaccine booster dosages, may be useful.

[0096] The composition can be employed in conjunction with a carrier,which may be any of a wide variety of carriers. Representative carriersinclude sterile water, saline, buffered solutions, mineral oil, alum,and synthetic polymers. Additional agents to improve suspendability anddispersion in solution may also be used. The selection of a suitablecarrier is dependent upon the manner in which the composition is to beadministered. The composition is generally employed in non-human animalsthat are susceptible to enzootic pneumonia, in particular, swine.

[0097] The composition may be administered by any suitable method, suchas intramuscular, subcutaneous, intraperitoneal or intravenousinjection. Alternatively, the composition may be administeredintranasally or orally, such as by mixing the active components withfeed or water, or providing a tablet form. Methods such as particlebombardment, microinjection, electroporation, calcium phosphatetransfection, liposomal transfection, and viral transfection areparticularly suitable for administering a nucleic acid. Nucleic acidcompositions and methods of their administration are known in the art,and are described in U.S. Pat. Nos. 5,836,905; 5,703,055; 5,589,466; and5,580,859, which are herein incorporated by reference. Other means foradministering the composition will be apparent to those skilled in theart from the teachings herein; accordingly, the scope of the inventionis not limited to a particular delivery form.

[0098] The composition may also include active components or adjuvants(e.g., Freund's incomplete adjuvant) in addition to the antigen(s) orfragments hereinabove described. Adjuvants may be used to enhance theimmunogenicity of an antigen. Among the adjuvants that may be used areoil and water emulsions, complete Freund's adjuvant, incomplete Freund'sadjuvant, Corynebacterium parvum, Hemophilus, Mycobacterium butyricum,aluminum hydroxide, dextran sulfate, iron oxide, sodium alginate,Bacto-Adjuvant, certain synthetic polymers such as poly amino acids andco-polymers of amino acids, saponin, iota carrageenan, Regressin™,Avridine™, Mannite monooleate, paraffin oil, and muramyl dipeptide.

[0099] Nucleic acid or polypeptide compositions or vaccines as describedherein can be combined with packaging materials including instructionsfor their use to be sold as articles of manufacture or kits. Componentsand methods for producing articles of manufactures are well known. Thearticles of manufacture may combine one or more vaccines (e.g., nucleicacid or polypeptide) as described herein. Instructions describing how avaccine is effective for preventing the incidence of a M. hyopneumoniaeinfection, preventing the occurrence of the clinical signs of a M.hyopneumoniae infection, ameliorating the clinical signs of a M.hyopneumoniae infection, lowering the risk of the clinical signs of a M.hyopneumoniae infection, lowering the occurrence of the clinical signsof a M. hyopneumoniae infection and/or spread of M. hyopneumoniaeinfections in animals may be included in such kits.

[0100] Conveniently, vaccines of the invention may be provided in apre-packaged form in quantities sufficient for a protective dose for asingle animal or for a pre-specified number of animals in, for example,sealed ampoules, capsules or cartridges.

[0101] Application of the teachings of the present invention to aspecific problem or environment is within the capabilities of one havingordinary skill in the art. Examples of the products and processes of thepresent invention appear in the following examples.

[0102] The invention will be further described in the followingexamples, which do not limit the scope of the invention described in theclaims.

EXAMPLES

[0103] A. P102 and Paralogs Thereof

Example A.1 Mycoplasma Strains

[0104]Mycoplasmas hyopneumoniae strains used included the 232, J, andBeaufort. The source and culture conditions used to grow M.hyopneumoniae are as described in Scarman et al. (1997) Microbiology143:663-673.

Example A.2 Cloning of the Gene Encoding P102

[0105] The gene encoding P102 was obtained by polymerase chain reaction(PCR) and cloned into pTrcHis (Invitrogen). The oligonucleotides TH130and TH131 were used to amplify the region encoding amino acids 33 to 887of P102 from pISM1217 as described in Hsu and Minion ((1998) Infect.Immun. 66:4762-4766). The PCR product having 5′ BamHI and 3′ PstIrestriction enzyme sites was digested sequentially with BamHI and PstI,gel purified, and ligated into BamHI/PstI-digested pTrcHis plasmid DNA.The ligation mixture was transformed into CSH50 Escherichia coli, andtransformants were selected for ampicillin resistance (100 μg per mL).The resulting plasmid was sequenced with primer SA1528 to confirm theinsertion and orientation of the insert.

[0106] Site directed mutagenesis was performed on the insert sequence toremove TGA codons, which code for tryptophan in Mycoplasmas. Directedmutagenesis was performed using the Stratagene QuikChange Site-DirectedMutagenesis Kit (Stratagene, CA) according to the manufacturer'sinstructions. Five TGA codons in the cloned sequence were changed to TGGusing the following primer pairs: P102.2f: 5′-GAT AAT TTT AAA AAA TGGTCG GCA AAA ACA GTT TTA (SEQ ID NO:21) ACT GCT GCC-3′; P102.2r: 5′-GGCAGC AGT TAA AAC TGT TTT TGC CGA CCA TTT TTT (SEQ ID NO:22) AAA ATTATC-3′; P102.3f: 5′-GAA AGA GGA AGT AAT TGG TTT TCA CGA CTT GAA AGA (SEQID NO:23) GC-3′; P102.3r: 5′-GCT CTT TCA AGT CGT GAA AAC CAA TTA CTT CCTCTT (SEQ ID NO:24) TC-3′; P102.4f: 5′-CTA AAA TTC TAA AAT CCT GGC TTGAAA CAA ATC TTC (SEQ ID NO:25) AAG GC-3′; P102.4r: 5′-GCC TTG AAG ATTTGT TTC AAG CCA GGA TTT TAG AAT (SEQ ID NO:26) TTT AG-3′; P102.5f:5′-GCC TCT CTG ATT ATT GGT ATG GAT CTC CGA ATT C-3′; (SEQ ID NO:27)P102.5r: 5′-GAA TTC GGA GAT CCA TAC CAA TAA TCA GAG AGG C-3′; (SEQ IDNO:28) P102.6f: 5′-GGG ACA AGC ATT TGG ACA GCT TTT AAT TTC G-3′; (SEQ IDNO:29) P102.6r: 5′-CGA AAT TAA AAG CTG TCC AAA TGC TTG TCC C-3′. (SEQ IDNO:30)

[0107]E. coli XL1-Blue MRF′ was the recipient for each mutagenesis step.To confirm the sequence and the single-base changes, and to determinewhether errors were introduced during the cloning and mutagenesis steps,the final product was sequenced using the primers: P102.2-SEQ: 5′-TCCGAC GAT GAC GAT AAG-3′; (SEQ ID NO:31) P102.5-SEQ: 5′-TGG AAA ATT AGTTCT TGG-3′; (SEQ ID NO:32) P102.6-SEQ: 5′-AGT TTC CAC TTC ATC GCC-3′.(SEQ ID NO:33)

[0108] The final construct was designated pISM1316.6.

Example A.3 Expression and Purification of P102

[0109] Plasmid pISM1316.6 was transformed into E. coli ER1458(F-Δ(lac)U169 lon100 hsdR araD139 rpsL(StrR) supF mcrAtrp+zjj202::Tn10(TetR) hsdR2(rk-mk+) mcrB1), a Lon protease mutant, inpreparation for protein expression. An overnight culture was diluted1:10 into fresh superbroth medium (per liter; 32 g Bacto tryptone, 20 gyeast extract, 5 g sodium chloride, pH 7.3) containing 1 mM isopropylthiogalactopyranoside (IPTG) and protease inhibitor cocktail (SigmaP8848) at a 1:200 dilution. The culture was incubated for 5 hours at 30°C. with shaking. The cells were collected by centrifugation andresuspended in TS buffer (10 mM Tris, 100 mM sodium chloride, pH 7.4)plus 8 M urea and 2 mg/mL of lysozyme. After incubating for 30 minuteson ice, the suspension was frozen in a dry ice ethanol bath and passedsequentially through three freeze-thaw cycles. The chromosomal DNA wassheared by passing the suspension through an 18-gauge needle, andinsoluble cellular debris was removed by centrifugation. The finalsolution was passed through a Talon Metal Affinity Resin (ClontechLaboratories, Inc., CA) column. The column was washed with 10 columnvolumes of TS buffer containing 10 mM imidazole. The bound protein waseluted with TS buffer containing 500 mM imidazole, and the column eluentwas dialyzed overnight against phosphate buffered saline (10 mM Na₂HPO₄,100 mM NaCl, pH 7.4). Purity of the protein preparations was assessed bysodium dodecyl sulfate gel electrophoresis and by Western blotting using6×His monoclonal antibody (Clontech).

Example A.4 Generation of P102 Antisera

[0110] Mice were immunized with 10 μg of purified P102 mixed with 200 μLof Freund's incomplete adjuvant, and on day 21, second dosages weregiven. Ascites were developed by the introduction of Sp2 myeloma cellsusing the method of Luo and Lin ((1997) BioTechniques 23:630-632), andascites fluid was aliquoted and stored at −70° C. Antibody specificitywas tested by immunoblot analysis using purified P102 protein and M.hyopneumoniae whole antigen.

Example A.5 Immunoelectron Microscopic Analysis of Immunogold-LabeledCell Sections

[0111] To determine if P102 is surface exposed or associated with theP97 cilium adhesin, monospecific polyclonal anti-P102 antiserum was usedin the following immunoelectron microscopic studies to determine thelocation of P102 in the Mycoplasma cell.

[0112]M. hyopneumoniae strains 90-1 and 60-3 were grown in modifiedFriis media (Friis (1971) Acta Vet. Scand. 12:69-79) until mid log phaseas described (Hsu et al. (1997) J. Bacteriol. 179:1317-1323). The cellswere pelleted by centrifugation and washed once with phosphate bufferedsaline (PBS) by centrifugation. Cells were resuspended in PBS and thenreacted with either anti-P102 ascite fluid diluted 1:50, or F1B6 cellculture supernatant (Zhang et al. (1995) Infect. Immun. 63:1013-1019)diluted 1:10, overnight at 4° C. The next day, cells were washed fivetimes with PBS and then reacted for 30 minutes at room temperature withgoat anti-mouse IgG+IgM labeled with 10 nm gold particles (EYLaboratories, Inc., San Mateo, Calif.) diluted 1:25. The cells were thenwashed five times with PBS and pelleted by centrifugation.

[0113] The final cell pellets were fixed with 3% glutaraldehyde in 0.1 Msodium cacodylate buffer (pH 7.2) at 4° C. overnight. The pellets werewashed three times, 15 minutes each time, with 0.1 M sodium cacodylatebuffer and post fixed with 1% osmium tetroxide in 0.1 M sodiumcacodylate buffer for 2 hours at room temperature. The pellets were thenwashed with distilled water, passed through an acetone series andembedded in Embed 812 and Araldite (Electron Microscopy Sciences, FortWashington, Pa.).

[0114] For tracheal sections, Mycoplasma-free pigs were inoculatedintratracheally with M. hyopneumoniae strain 232 as described in Thackeret al. ((1997) Potentiation of PRRSV pneumonia by dual infection withMycoplasma hyopneumoniae. In Conference of Research Workers in AnimalDiseases. Ellis, R. P. (ed.) Chicago, Ill.: Iowa State University Press,pp. 190). At 10 and 21 days, pigs were sacrificed, and tracheas wereremoved. One cm blocks of tissue were fixed with 1% glutaraldehydeovernight, dehydrated in an acetone series and embedded as above. Thick(1-2 μm) sections were stained with methylene blue polychrome andexamined by microscopy for regions containing ciliated epithelium. Thinsections (80-90 nm) were then prepared for labeling. For some studies,cells grown in vitro were embedded and sectioned prior to staining. Thesections were pretreated with ammonium chloride (1%) for 1 hour, 0.05 Mglycine in PBS for 15 minutes, and blocked for 30 minutes in 2% fishgelatin+2% bovine serum albumin in TS buffer (10 mM Tris, 100 mM NaCl,pH 7.5). Primary antibodies were diluted (1:50) in TS buffer and reactedwith sections for 30 minutes at room temperature. The sections werewashed six times with TS buffer, and then incubated with goat anti-mouseIgG+IgM labeled with 10 nm gold particles (diluted 1:2) for 15 minutesat room temperature. Both primary antibodies and the conjugate werediluted and centrifuged briefly (12,000×g for 5 minutes) to remove goldaggregates prior to use. The sections were then washed six times with TSbuffer, dried, contrasted with osmium vapors for 2 minutes, and stainedwith uranyl acetate-lead citrate. The sections were examined on aHitachi 500 electron microscope at 75 kV.

[0115] In in vitro grown cells, gold particles were found external tothe cells and were primarily associated with the extracellular matrix.Similar results were observed for cells that were stained before orafter fixation and sectioning. Occasionally, particles were seenassociated with the cell surface, and in rare cases, particles were seenintracellularly. In cells associated with swine cilia, however, goldparticles were seen at high concentration intracellularly. P102 was alsofound in association with swine cilia, often in aggregates or at highconcentrations. The extracellular matrix that was so prominent in brothgrown cells was not evident in sections of infected swine epithelia.

Example A.6 Two-Dimensional Electrophoresis

[0116] Two-dimensional gel electrophoresis (2-DGE) was carried outessentially as described by Guerreiro et al. ((1997) Mol. Plant MicrobeInteract., 10:506-16). First dimension immobilized pH gradient (IPG)strips (180 mm, linear and non-linear pH 3-10 and linear pH 4-7 and6-11; Amersham Pharmacia Biotech, Uppsala, Sweden) were prepared forfocusing by submersion in hydration buffer (8 M urea, 0.5% wt/vol CHAPS,0.2% wt/vol DTT, 0.52% wt/vol Bio-Lyte and a trace of bromophenol blue)overnight. M. hyopneumoniae whole cell protein (100 μg for analyticalgels, 0.5-1.0 mg for preparative gels and immunoblots) was diluted withsample buffer (8 M urea, 4% w/v CHAPS, 1% w/v DTT, 0.8% w/v Bio-Lyte3-10, 35 mM Tris, and 0.02% w/v bromophenol blue) to a volume of 50 to100 μL for application to the anodic end of each IPG strip. Isoelectricfocusing was performed with a Multiphor II electrophoresis unit(Pharmacia) for 200 kVh at 20° C. except for pH 6-11 strips, which wereelectrophoresed for 85 kVh. IEF strips were reduced and alkylated inTris-HCl (0.5 M, pH 6.8) containing 6 M urea, 30% w/v glycerol, 2% w/vsodium dodecyl sulfate (SDS), 2% w/v DTT and 0.02% bromophenol blue.Equilibrated strips were placed onto Pharmacia ExcelGels (T=12 to 14%acrylamide) for SDS-PAGE using the Multiphor II. Electrophoreticconditions consisted of 200 Volts for 1.5 hours followed by 4 hours at600 Volts at 5° C. Gels were stained in Coomassie Blue R-250 (Bio-Rad,Hercules, Calif.), and proteins were transferred to polyvinylidenedifluoride (PVDF) membranes using a Hoefer TE70 Series SemiPhor Semi-DryTransfer Unit (Amersham Pharmacia Biotech, Uppsala, Sweden). Thetransfer was carried out for 1.5 hours at maximum voltage and a currentmeasured by multiplying the area of the gel (cm²) by 0.8 mA.

Example A.7 Post-Separation Analyses

[0117] Protein spots were excised from gels using a sterile scalpel andplaced in a 96 well tray. Gel pieces were washed with 50 mM ammoniumbicarbonate/100% acetonitrile (60:40 v/v) and then dried in a Speed Vac(Savant Instruments, Holbrook, N.Y.) for 25 minutes. Gel pieces werethen hydrated in 12 μL of 12 ng μL⁻¹ sequencing grade modified trypsin(Promega, Madison, Wis.) for 1 hour at 4° C. Excess trypsin solution wasremoved and the gel pieces immersed in 50 mM ammonium bicarbonate andincubated overnight at 37° C. Eluted peptides were concentrated anddesalted using C₁₈ Zip-Tips™ (Millipore Corp., Bedford, Mass.). Thepeptides were washed on column with 10 μL of 5% formic acid. The boundpeptides were eluted from the Zip-Tip™ in matrix solution (10 mg mL⁻¹α-cyano-4-hydroxycinnamic acid [Sigma] in 70% acetonitrile) directlyonto the target plate. Matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry (MALDI-TOF MS) mass spectra wereacquired using either a PerSeptive Biosytems Voyager DE-STR (Framingham,Mass.) or a Micromass TofSpec2E (Micromass, Manchester UK). Bothinstruments were equipped with 337 nm nitrogen lasers. All spectra wereobtained in reflectron/delayed extraction mode, averaging 256 lasershots per sample. Two-point internal calibration of spectra wasperformed based upon internal porcine trypsin autolysis peptides (842.5and 2211.10 [M+H]⁺ ions). A list of monoisotopic peaks corresponding tothe mass of generated tryptic peptides was used to search a modifiedtranslated version of the M. hyopneumoniae genome. Successfulidentifications were based on the number of matching peptide masses andthe percentage sequence coverage afforded by those matches. N-terminalEdman sequencing was performed as previously described (Nouwens et al.,2000).

Example A.8 P102 is Surface Expressed

[0118] To generate a P102 specific antibody, recombinant P102 proteinwas expressed in in E. coli and then purified as follows. The codingsequence for P102 was obtained from plasmid pISM1217, which containedthe entire sequence of P102 (Hsu and Minion (1998) Infect. Immun.66:4762-4766). The region of the coding sequence encoding amino acids33-887 was amplified by PCR using primers having BamHI and PstIrestriction sites at the 5′ termini to enable cloning into pTrcHis. Theresulting construct was designated pISM1249. To allow for expression ofthe coding sequence in E. coli, the TGA codons in the pISM1249 sequencewere altered by site-directed mutagenesis to TGG codons. The finalconstruct pISM1316.6 was sequenced to confirm these changes and to checkfor errors introduced by PCR during the mutagenesis step.

[0119] Expression of the cloned sequence in pISM1316.6 resulted in apoly-histidine-tagged protein of about 100 kDa. Expression levels ofP102 were low in E. coli despite the removal of the opal (TGA) stopcodons. A Talon Metal Affinity Resin column was used to removecontaminating E. coli proteins during purification. Mouse hyperimmuneantiserum raised against this recombinant protein was used in immunoblotanalysis of M. hyopneumoniae whole cells. The anti-P102 antiserum showedthree bands indicating either the presence of cross-reactive proteins orthat P102 was being proteolytically processed. Trypsin treatment ofwhole cells followed by immunoblot and development with the anti-P102antiserum showed that P102 was located on the membrane surface; allimmunoreactive bands were sensitive to trypsin.

Example A.9 P102 Paralogs are Found Throughout the M. hyopneumoniaeGenome

[0120] Hybridization studies indicated that P102 or P102-relatedsequences may exist in multiple copies in the genome of M. hyopneumoniae(Hsu et al. (1997) J. Bacteriol. 179:1317-1323). Genome sequencingstudies have identified four distinct paralogs of P102 (C2-mhp210,C27-mhp348, C28-mhp663, and C2-mhp036) and two partial paralogs(C2-mhp033 and C2-mhp034) scattered throughout the chromosome (FIG. 21).Further analysis of the genome sequence of M. hyopneumoniae revealedadditional open reading frames with varying homologies to P102. Each ofthese appeared to be a fusion with a second gene, while the originalP102 sequence had undergone significant evolution. Also, each paralogwas part of a two-gene genetic structure, possibly organized intooperons. In every case, the P102 paralog was the second or downstreamgene. DNA sequence analysis of each of the P102 paralogs showed thathomology to P102 was low, but amino acid homology was much higher. Theamino acid sequences of the P102 paralogs are shown in FIGS. 2, 6, 12,14, 16,18, and 20.

Example A.10 Biotin Labeling of Surface Accessible Proteins IdentifiedMolecules Belonging to a Multi-Gene Family

[0121] Studies were undertaken to identify all of the surface accessibleproteins in M. hyopneumoniae recognized by convalescent and hyperimmuneswine sera. By combining surface biotinylation, two-dimensionalimmunoblotting, genomic and proteomic analysis, a subset of thesesurface molecules was mapped to the genome sequence of M. hyopneumoniae.

[0122] Initially, two-dimensional gel electrophoresis of biotinylatedproteins identified groups of proteins that were surface exposed, highlyexpressed, and appeared to resolve along the pI gradient as a series ofspots. The molecular masses of many of these proteins ranged from 40 to130 kDa. Many of these proteins were recognized by convalescent andhyperimmune swine sera. This suggests that these proteins were expressedduring M. hyopneumoniae infection and evoked an accompanying immuneresponse.

[0123] Tryptic fragments of individual protein spots were analyzed bypeptide mass fingerprinting, and the spectra matched to theoreticaltrypsin cleavage products generated from the M. hyopneumoniae genomedatabase. Some of the spots of different molecular masses mapped to thesame single copy gene.

Example A.11 Peptide Mass Fingerprinting and Biotinylation Studies ShowThat P102 Paralogs are Expressed

[0124] Many of the proteins identified by biotinylation and peptide massfingerprinting were related to products from the cilium adhesion operon(Hsu and Minion (1998) Infect. Immun. 66:4762-4766). In addition to thecilium adhesin P97, gene products representing P102 and related proteinswere identified.

A.12 Results

[0125] Results indicated that there were a surprising number of P102paralogs that were all expressed and located on the surface of theorganism. Some of the P102 paralogs had a greater degree of sequenceidentity with P97, while other P102 paralogs did not. None of thesequences surrounding the P102 paralogs were similar, which suggeststhat the P102 genes duplicated and moved independently of surroundingsequences. Differential staining of in vitro-grown and in vivo-grownorganisms was observed, further suggesting that P102 might be involvedin the hyperimmune-like responses seen during infection.

[0126] B. P216 Studies

Example B.1 Mycoplasma Strains and Culture

[0127] The source and culture conditions used to grow M. hyopneumoniaestrains J, Beaufort and 232 are as described in Scarman et al. ((1997)Microbiology 143:663-673). Mycoplasmas were harvested by centrifugationat 10,000×g, washed three times with TS buffer (10 mM Tris, 150 mM NaCl,pH 7.5), and the final cell pellets were frozen at −20° C. until use.

Example B.2 Preparative Electrophoresis

[0128] Preliminary vaccine trials in swine immunised withsize-fractionated antigens of M. hyopneumoniae indicated that antigenpools residing in two fractions, fractions 2 (85-150 kDa) and 3 (70-85kDa), provided limited protection against a virulent challenge(Djordjevic et. al (1997) Aust Vet J 75:504-511). To determine the aminoacid sequences of proteins residing in these molecular mass fractions,whole cell lysates of M. hyopneumoniae J strain were separated using5-7% polyacrylamide resolving columns each with a 4% stacking gel usinga BioRad 491 Prep Cell as described in Scarman et al. ((1997)Microbiology 143:663-673). Proteins corresponding to those defined forfractions 2 and 3 were pooled, concentrated by filtration, andresuspended in PBS. Protein fractions were digested with trypsin,separated using electrophoresis on precast 8-15% gradient Tricine gels(Novex), and then blotted onto PVDF membrane (BioRad, California, USA)(Towbin et al. (1979) Proc. Natl. Acad. Sci. USA. 76:4350-4354). Proteinfractions were analyzed by (1) reaction with porcine hyperimmune seraraised against the J strain of M. hyopneumoniae and (2) staining withamido black. Tryptic fragments stained with amido black that reactedwith the hyperimmune sera were analysed by N-terminal amino acidsequencing.

Example B.3 Cloning of the Gene Encoding P216

[0129] To clone the genes encoding immunoreactive proteins, degenerateoligonucleotide probes were designed from the N-terminal peptidesequences determined above and used to probe EcoRI-digested chromosomalDNA by Southern analysis (Southern (1975) J. Mol. Biol. 98:503-517).EcoRI digested chromosomal DNA from the Beaufort strain was separated ona 1% agarose column prepared in 491 Prep Cell according to the BioRadTechnical Note #2203. Samples from every fifth fraction were blotted toa nylon membrane and probed with degenerate oligonucleotide probesderived from the N-terminal sequences of tryptic fragments. DNAfragments from reactive fractions were incubated with the Klenowfragment and Pfu DNA polymerase to generate blunt ends. DNA fragmentswere ligated into pCR Script™ and transformed into XL10-Gold as outlinedin the manufacturer's instructions (Stratagene).

[0130] In this way, N-terminal sequence analysis of an X kDa trypticpeptide fragment recognised by porcine hyperimmune generated thesequence ELEDNTKLIAPNIRQ (SEQ ID NO:34). Based on this amino acidsequence, a degenerate oligonucleotide having the sequence 5′-GAA(T/C)T(T/A) GAA GAT AAT AC(C/A/T) AAA TTA ATT GC(T/A) CCT AAT-3′ (SEQ IDNO:35) was made and used as a probe to identify a hybridizing fragmentof 4.5 kb. The clone containing this 4.5 kilobase fragment wasdesignated p216.

Example B.4 DNA Sequence Analysis

[0131] For sequence analysis, purified plasmid DNA (Qiagen) or PCRproduct purified from agarose using the BRESA-CLEAN™ kit (Bresatec,Adelaide, Australia) was used. Oligonucleotide primers were obtainedcommercially (Sigma), and the BigDye™ Terminator Cycle Sequencing Kit(Applied Biosystems) was used for sequencing reactions. Results wereanalysed with an Applied Biosystems Model 377 automated sequencer.

[0132] Sequence analysis of the cloned fragment in p216 from theBeaufort strain revealed a large ORF that did not significantly matchsequences deposited in GenBank. The fragment was the carboxy terminus ofa larger ORF as the fragment had a stop codon but no ATG start codon.Additional upstream sequence was obtained by inverse PCR, and the finalN-terminal sequence was obtained by PCR using primers designed fromstrain 232 genomic sequences. The complete ORF (C28-mph545; see, FIG. 7)was 5,637 base pairs in length and encoded a protein of 216 kDadesignated P216 (C28-MPH545; see, FIG. 8). The ORF contained 17 TGAcodons, 12 of which appeared in the carboxy terminal 85 kDa.

[0133] Blastp analysis of the complete gene sequence revealed nearidentity with the partial gene sequence YX2 (GenBank Accession No.AF279292) from M. hyopneumoniae strain 232 and limited sequence homologywith the P97 cilium adhesin (GenBank Accession No. U50901) with 21%identities, 38% positives and 19% gaps (Expect=4e-18). Comparisons ofthe nucleotide and derived protein sequences with the database wereperformed using the package from the University of Wisconsin GeneticsGroup (GCG) Version 7, accessed via the Australian National GenomicInformation Service (ANGIS, University of Sydney) and MacVector(Scientific Imaging Systems, Eastman Kodak Co., New Haven, Conn.).

[0134] DNA sequence encoding the P216 homologue from the 232 strain ofM. hyopneumoniae was obtained as part of a genome-sequencing project.Southern blotting analysis using an oligonucleotide probe from thecarboxy terminus showed that the M. hyopneumoniae genome contained asingle copy of the gene encoding the 216-kDa protein. Blastn analysiswith p216 and the M. hyopneumoniae genome database also identified asingle copy. The protein has 1,879 amino acids, a pI of 8.51, and ishighly hydrophilic. A protein motif search using the algorithm Prositeon the ISREC Profilescan server(www.isrec.isb-sib.ch/software/PFSCAN_form.html) identified a bipartitenuclear binding domain (BNBD) between amino acids 1012-1029.

[0135] The nucleotide sequence of the M. hyopneumoniae p216 gene fromstrain 232 and the J strain are shown in FIGS. 7 and 19, respectively.

Example B.5 Generation of Antisera Against M. hyopneumoniae Strain 232

[0136] Preparation of porcine hyperimmune serum against M. hyopneumoniaeis as described in Scarman et al. (1997) Microbiology 143:663-673. Inbrief, M. hyopneumoniae-free swines were challenged with a preparationof M. hyopneumoniae strain 232 emulsified in Freund's complete adjuvant,and these swines were subjected to a second exposure one month laterwith the same preparation in Freund's incomplete adjuvant. Serumresponses were monitored until an anti-M. hyopneumoniae response wasconfirmed by an enzyme-linked immunosorbent assay (ELISA).

Example B.6 Generation of P216 Polyclonal Antisera

[0137] To generate monospecific polyclonal antisera to P216, the DNAsequence encoding P216 from strain 232 was examined for the presence ofTGA codons, since TGA codons encode tryptophans in Mycoplasmas. A regioncontaining no TGA codons and encoding a 30 kDa protein (amino acids1043-1226) was identified. PCR primers were designed to amplify andclone this region into pCR Script™ forming plasmid p216.1. The clonedfragment was then directionally cloned into pQE9 (Qiagen) by ligation ofBamHI- and HindIII-digested p216.1 DNA to form p216.2. The ligationmixture was transformed into Escherichia coli M15[pREP4] according tothe manufacturer's instructions (Qiagen). Colony hybridization using theDIG system (Roche) was used to identify transformants containing theproper fragment.

[0138] Cultures of the transformants containing p216.2 were grown in LBmedium (Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual,2^(nd) Ed, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.)containing ampicillin (100 μg/mL) and kanamycin (25 μg/mL) at 37° C.with shaking. For expression from p216.2, cultures were treated with 1mM isopropyl-β-D-thiogalactopyranoside (IPTG) after reaching an OD₆₀₀ of0.6. After induction for 4 hours, the cells were harvested bycentrifugation at 4,000×g for 20 minutes. Purification of therecombinant His-tagged protein was achieved using Ni-NTA resin underdenaturing conditions as outlined in the manufacturer's instructions(Qiagen).

[0139] Purified recombinant protein was dialysed against PBS containing5% glycerol and concentrated using polyvinyl-pyrrolidone (Sigma).Approximately 5 mg of purified protein in a volume of 250 μL wereemulsified with an equal volume of Freund's incomplete adjuvant (Sigma).The preparation was given subcutaneously to rabbits at two sites and abooster immunization, similarly prepared, was given three weeks later.Serum response against the immunizing antigen was confirmed byimmunoblot analysis.

[0140] Similarly, rabbit antisera directed against the N-terminalsequence of P216 were generated by immunization with the peptideDFLTNNGRTVLE (SEQ ID NO:36) (amino acids 94-105 of P216) conjugated tokeyhole limpet hemocyanin. Rabbit immunizations were performed asdescribed in (Scarman et al. (1997) Microbiology 143:663-673).

Example B.7 Electrophoretic and Immunoblot Analyses

[0141] Sodium dodecyl sulphate polyacrylamide gel electrophoresis(SDS-PAGE) and immunoblot analysis were performed as described byLaemmli (1970) Nature 227:680-685 and Towbin et al. (1979) Proc. Natl.Acad. Sci. USA, 76:4350-4354, respectively. Analytical electrophoreticgels containing M. hyopneumoniae strain 232 proteins were stained withsilver (Rabilloud et al. (1992) Electrophoresis 13:264-266). Preparativegels were stained with colloidal Coomassie Brilliant Blue G-250 (0.1%Coomassie Brilliant Blue G-250 w/v, 17% w/v ammonium sulfate, 34%methanol v/v, 3% v/v ortho-phosphoric acid). Gels were destained in 1%v/v acetic acid for 1 hour.

[0142] Immunoblot analysis was used to determine if P216 is recognisedby antibodies elicited during natural infection using swine field serashown to contain antibodies against M. hyopneumoniae (Djordjevic et al.(1994) Vet. Microbiol. 39:261-273). The 30 kDa recombinant proteinrepresenting amino acids 1043-1226 of P216 was used as antigen in theseexperiments. Other immunoblot analyses included one- and two-dimensionalblots of M. hyopneumoniae whole cells using swine convalescent serapools (2D blots) and individual swine sera (1D blots). Swine hyperimmunesera were also used to screen for immunoreactive proteins in one- andtwo-dimensional immunoblot analyses. Rabbit antisera generated againstthe 30 kDa recombinant protein and the peptide DFLTNNGRTVLE (SEQ IDNO:36) specific for P130 were used to investigate processing of P216 inone-dimensional immunoblotting experiments as well.

Example B.8 Two-Dimensional Gel Electrophoresis

[0143] Two-dimensional gel electrophoresis was carried out essentiallyas described by Guerreiro et al. ((1997) Mol Plant Microbe Interact10:506-516). First dimension immobilized pH gradient (IPG) strips (180mm, linear and non-linear pH 3-10 and linear pH 4-7;Pharmacia-Biotechnology, Uppsala, Sweden) were prepared for focusing bysubmersion in rehydration buffer (8 M urea, 0.5% w/v CHAPS, 0.2% w/vDTT, 0.52% w/v Bio-Lyte and a trace of bromophenol) overnight. M.hyopneumoniae 232 whole cell proteins (100 μg for analytical gels,0.5-1.0 mg for preparative gels and immunoblots) were diluted withsample buffer (8 M urea, 4% w/v CHAPS, 1% w/v DTT, 0.8% w/v Bio-Lyte3-10, 35 mM Tris, and 0.02% w/v bromophenol blue) to a volume of 50 to100 μl for application to the anodic end of each IPG strip. Isoelectricfocusing was run with the Immobiline DryStrip kit in a Multiphor IIelectrophoresis unit (Pharmacia-Biotechnology) for 200 kVh at 20° C. IEFstrips were subsequently prepared for second dimensionSDS-polyacrylamide gel electrophoresis (SDS-PAGE) by equilibration inTris-HCl (0.5 M, pH 6.8) containing 6 M urea, 30% w/v glycerol, 2% w/vsodium dodecyl sulfate (SDS), 2% w/v DTT, and 0.02% bromophenol blue.Equilibrated strips were placed onto Pharmacia ExcelGel gels (T=12 to14% acrylamide) for molecular mass separation of M. hyopneumoniaeproteins on a Multiphor II unit. Electrophoretic conditions consisted of200 Volts for 1.5 hour followed by 4 hours at 600 Volts. Gels weremaintained at 5° C. throughout.

Example B.9 Peptide Mass Fingerprinting-Mass Spectrometry

[0144] Proteins spots were manually excised and placed in a 96-wellmicrotiter plate. Conditions used for trypsin digestion and for thegeneration of peptide mass fingerprints are described in Nouwens et al.(2000) Electrophoresis 21:3797-3809. A purification step was performedon the tryptic peptides for proteins with poor peptide mass fingerprintsas described in Gobom et al. (1999) J. Mass Spectrom. 34:105-116.Protein identifications were assigned by comparing the peak listsgenerated from peptide mass fingerprinting data to a database containingtheoretical tryptic digests of M. hyopneumoniae strain 232. TheProtein-Lynx package (Micromass, Manchester, UK) was used to searchdatabases.

Example B.10 Image Processing

[0145] Gels and immunoblots were digitized at 600 dpi with a UMAXPS-2400X lamp scanner using Photoshop 3.0 (Adobe, Mountain View,Calif.). Spot detection and gel-to-gel protein spot matching wereperformed with MELANIE II software (BioRad, Hercules, Calif.) run underOpenWindows 3.0. Apparent molecular masses were determined byco-electrophoresis with protein standards (Pharmacia-Biotechnology).

Example B.11 Results of Two-Dimensional Electrophoresis and Peptide MassFingerprinting Analysis

[0146] Analyses of two-dimensional electropherograms identified twoclusters of spots that tracked along the pI gradient in an unusualfashion. Peptide mass fingerprinting analysis of spots within each ofthe clusters showed that the spots had identical mass fingerprints andwere thus derived from the same molecule. Cluster 1 with an approximatemass of 130 kDa was mapped to the N-terminal region of P216 from thegenome sequence of M. hyopneumoniae strain 232. Cluster 2 ofapproximately 85 kDa mapped to the carboxy terminus of the same ORF. Theproteins were designated P130 and P85, respectively. The pI of cluster 1ranged from 9.5 to 8.0, while the p1 of cluster 2 ranged from 9.0 to6.5. Mass spectrometric analysis indicated that P216 was cleaved betweenamino acids 1004 and 1090 generating the two fragments of 130 and 85kDa.

Example B.12 Results of Immunoblot Analysis

[0147] Two-dimensional immunoblots reacted with porcine hyperimmune serarevealed a complex pattern of spots two of which corresponded to P130and P85. P85 was also strongly recognized by a pool of convalescent serashowing that it was an important antigen during disease. To investigatethis further, a 30-kDa region spanning amino acids 1042-1226 in P85 wasexpressed, purified by nickel-affinity chromatography, and blotted ontoPVDF membrane. Individual convalescent sera from swines known to bepositive in a M. hyopneumoniae-specific ELISA reacted with the 30-kDaprotein confirming that P216 is an important molecule recognized by thehost immune response during the normal course of infection. Antibodiesraised to a 30-kDa peptide spanning amino acids 1042-1226 reacted solelywith the 85 kDa cleavage product suggesting that cleavage occurredbetween amino acids 1004 and 1042. Sera raised to the N-terminal peptideof P216 recognized only P130

Example B.13 Posttranslational Processing of P216 Among DifferentStrains of M. hyopneumoniae

[0148] To investigate fragment patterns of P216 in different M.hyopneumoniae strains, immunoblot analysis was performed with theanti-P130 N-terminal peptide and anti-P30 antisera. Antibodies raisedagainst the N-terminal peptide recognized P130 and several lowermolecular mass peptides in one-dimensional immunoblots of whole celllysates of J and 232 strains. The pattern of proteins recognised by thisantisera was different between the two strains. Antisera raised againstthe 30-kDa peptide strongly recognised an 85-kDa antigen in both J and232 strains, but also reacted with a number of weakly reactive proteins.Similarly, the pattern recognised with the anti-30-kDa sera wasdifferent between J and 232.

[0149] To determine if different post-translational cleavage events wereoccurring among other strains of M. hyopneumoniae, a collection ofstrains from different geographic origins were examined by immunoblot.Anti-30 kDa sera reacted strongly to an 85-kDa antigen and otherproteins of lower molecular mass in immunoblots of whole cell lysatesfrom different strains of M. hyopneumoniae. These strains representedisolates recovered from different geographic locations within Australiaand from different countries including the USA, Great Britain andFrance. The anti-P30 sera, however, did not react against antigens inimmunoblots of whole cell lysates of related porcine Mycoplasmas, e.g.Mycoplasma hyorhinis and Mycoplasma flocculare, suggesting that P216 isa M. hyopneumoniae-specific antigen. Convalescent sera from differentswines also recognized purified recombinant P30 indicating that P216 isexpressed in vivo.

Example B.14 Surface Localization Studies

[0150] Several approaches were taken to determine if P216 and itscleavage products were associated with the outer membrane surface. Theseincluded trypsin digestion and cell surface biotinylation.

[0151] For trypsin digestion studies, all solutions and M. hyopneumoniaecell stocks were pre-equilibrated at 37° C. M. hyopneumoniae cells (200mg/mL in PBS) were aliquoted (300 μL) into sterile eppendorf tubes at37° C. and trypsin was added to a final concentration ranging from0.1-1000 μg/mL. The suspensions were inverted gently and incubated at37° C. for 20 minutes. Immediately after incubation, the cells werelysed in Laemmli buffer, heated at 95° C. for 10 minutes and analysed bySDS PAGE and immunoblotting. Trypsin digested both P85 and P130 in aconcentration dependent manner, but did not digest the intracellularenzyme lactate dehydrogenase, a control for spontaneous lysis of cells(Strasser et al. (1991) Infect. Immun. 59:1217-22). This suggests thatboth portions of P216 are surface accessible and sensitive to trypsindigestion.

[0152] To further clarify this, surface biotinylation of M.hyopneumoniae was performed. The method described by Meier et al.((1992) Anal. Biochem. 204:220-226) was used with the followingmodifications. All solutions were pre-chilled at 4° C. and allmanipulations were performed on ice. M. hyopneumoniae pellets (200 mgwet weight) were resuspended in 4 mL of BOS buffer (10 mM sodiumtetraborate in 0.15 M NaCl, pH 8.8). Immediately after the addition of 5μL of NHS-biotin (10 mg/mL in dimethylsulfoxide), the reaction wasallowed to proceed for 1 to 8 minutes with swirling. To determine themost suitable reaction time, aliquots were removed at 1-minute intervalsfor 15 minutes. A reaction time of 5 minute was chosen for allsubsequent studies except where noted. Biotinylation was stopped withthe addition of 2 mL of 0.1 M NH₄Cl that served to saturate unboundNHS-biotin. Cells were harvested by centrifugation (8,500×g, 10 minutes)and washed twice in TKMS buffer (25 mM Tris-HCl, pH 7.4, 25 mM KCl, 5 mMMgCl₂ and 0.15 M NaCl in PBS). The products were resolved bytwo-dimensional electrophoresis.

[0153] Both P130 and P85 were readily biotinylated, confirming that allparts of P216 were surface accessible.

Example B.15 Triton X-100 and X-114 Extractions

[0154] Integral membrane proteins from 200 mg wet weight of whole cellswere extracted with TX-114 essentially as described by Bordier ((1981)J. Biol. Chem. 182:1356-1363). The resultant aqueous and detergentphases were collected and analysed by SDS-PAGE and immunoblotting. Thephase partitioning activity of Triton X-114 causes separation ofhydrophobic molecules into the detergent phase. When treated with TritonX-114, P85 remained in the insoluble pellet consisting of complex highmolecular weight structures that (1) were membrane associated and (2)lacked the solubility of normal cytosolic proteins.

[0155] For Triton X-100 extraction, pelleted M. hyopneumoniae (strains Jand Beaufort) cells (200 mg wet weight) were resuspended in 10 mL of TSbuffer containing 1 mM phenylmethylsulfonyl fluoride. Proteins wereextracted by the addition of 2% Triton X-100 (Amersham PharmaciaBiotechnology) and incubated at 37° C. for 30 minutes as described inStevens and Krause ((1991) J. Bacteriol 173:1041-1050). Briefly, M.hyopneumoniae cell suspensions were centrifuged (14,000×g, 30 min) at 4°C. The aqueous phase was removed and the pellet was re-extracted asdescribed above. The insoluble pellet and both aqueous phases wereanalysed by SDS-PAGE and immunoblotting using anti-30 kDa and seraraised against the peptide DFLTNNGRTVLE (SEQ ID NO:36).

[0156] With Triton X-100 fractionation, high molecular weightcytoskeletal-like proteins remain insoluble, but phase partitioning doesnot occur. When treated with Triton X-100, P85 partitioned primarily tothe aqueous detergent-containing phase, but about 30% remained in thepellet. These data indicate that P216 may form extracellular oligomericstructures. The presence of coiled coil domains in both fragments ofP216 also supports this hypothesis.

[0157] C. P97 Studies

Example C.1 Bacterial Strains and Plasmids

[0158]M. hyopneumoniae strains 232 (virulent parental strain),232_(—)91.3 (high adherent clone), 232_(—)60.3 (low adherent clone), andJ type strain (NCTC 10110) were grown in modified Friis broth andharvested as described by Zhang et al. ((1995) Infect Immun63:1013-1019) and Djordjevic et al. ((1994) Vet Microbiol 39:261-273),respectively. All broth media were filter sterilized through 0.22 μmfilters, which removed the majority of particulate matter. Mycoplasmaswere harvested by centrifugation and extensively washed to removeremaining medium contaminants. Escherichia coli TOP10 containing pISM405was grown on Luria Bertani (LB) agar or in LB broth (Sambrook et al.,1989) containing 100 μg ml⁻¹ ampicillin.Isopropyl-β-D-thiogalactopyranoside (IPTG) induction was carried out bythe addition of IPTG to a final concentration of 1 mM. Bacterialcultures were routinely grown at 37° C. and liquid cultures were aeratedby shaking at 200 rpm.

Example C.2 Construction and Expression of Adhesin Fusion Protein

[0159] Hexa-histidyl P97 fusion proteins were constructed using thepTrcHis (Invitrogen, Carlsbad, Calif.) cloning vector. Primers FMhp3(5′-GAA CAA TTT GAT CAC AAG ATC CTG AAT ATA CC-3′ (SEQ ID NO:37)) andRMhp4 (5′-AAT TCC TCT GAT CAT TAT TTA GAT TTT AAT TCC TG-3′ (SEQ IDNO:38)) were used to amplify a 3013 bp fragment representing base pairs315-3321 of the gene sequence containing amino acids 105-1107. Thefragment was digested with BclI (underlined sequence) and inserted intothe BamHI site of vector pTrcHisA. A construct with the proper fragmentorientation was identified by restriction digests. The resulting 116-kDarecombinant P97-polyhistidine fusion protein contained the R1 and R2repeat regions as well as the major cleavage site at amino acid 195 inthe P97 sequence.

Example C.3 Antisera

[0160] The Mab F1B6 has been described (Zhang et al. (1995) Infect.Immun. 63:1013-1019). Mab F1B6 binds to the R1 region of the ciliumadhesin that has at least 3 repeat sequences (Minion et al. (2000)Infect. Immun. 68:3056-3060). Peptides with sequences TSSQKDPST (ΔNP97)(SEQ ID NO:39) and VNQNFKVKFQAL (NP97) (SEQ ID NO:40) were used to raiseantibodies against P97/P66 and P22, respectively. The peptides werebound to keyhole limpet hemocyanin with the Pierce Imjet MaleimideActivated Immunogen Conjugation Kit (Pierce Chemical Co., Rockford,Ill.). These conjugates were then used to generate mouse hyperimmuneantisera by the method of Luo and Lin ((1997) BioTechniques 23:630-632).The resulting antisera were tested by enzyme linked immunosorbent assay(ELISA) using ovalbumin-peptide conjugate and purified recombinant P97antigens, and by immunoblot with the recombinant P97 antigen. Antiserumraised against the C-terminal 28 kDa (R2 serum) of the cilium adhesin ofstrain J has been described (Wilton et al. (1998) Microbiology144:1931-1943). Mouse Mab 2B6-D4 raised against human fibronectin waspurchased commercially (BD Biosciences, Pharmingen) as was alkalinephosphatase conjugated goat anti-mouse Ig(H+L) antibodies (SouthernBiotechnology Associates, Inc., Birmingham, Ala.). Goat anti-mouseIgG+IgM labeled with 10 nm colloidal gold particles (EY Laboratories,Inc., San Mateo, Calif.) was used in immunogold electron microscopystudies.

Example C.4 Immunoblot Analysis

[0161] Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) and immunoblot analysis was performed as described by Laemmli((1970) Nature 227:680-685) and Towbin et al. ((1979) Proc. Natl. Acad.Sci. USA. 76:4350-4354), respectively. Proteins were transferred to PVDFmembranes (Micron Separations, Inc.). For the media control experiments,purified recombinant P97 was incubated with fresh and spent Friis media.Spent media was prepared from an early log phase culture that had beencentrifuged and filtered through a 0.1 μm filter. Purified recombinantP97 (2.5 μg) in 20 μl phosphate buffered saline was diluted 1:1 in freshor spent media and incubated overnight at 37° C. Ten μl of the mixturewere the loaded onto SDS-PAGE gels, blotted to nitrocellulose anddeveloped with F1B6 Mab. For ligand blotting, PVDF blots weretransferred, blocked and washed as described previously (Wilton et al.(1998) Microbiology 144:1931-1943). Blots were exposed to humanfibronectin (5 μg ml⁻¹) dissolved in TS buffer (TS buffer: 10 mMTris-HCl, pH 7.4; 150 mM NaCl) for 1.5 h, washed, and exposed to 0.4 μgml⁻¹ anti-human fibronectin Mabs for 1 h at room temperature. Blots werewashed and developed as described above.

Example C.5 Trypsin Treatment of M. hyopneumoniae

[0162]M. hyopneumoniae cells (0.5 g) were treated with trypsinessentially as described previously (Wilton et al. (1998) Microbiology144:1931-1943). Briefly, trypsin was added to cell suspensions of M.hyopneumoniae at 0, 0.3, 0.5, 1.0, 3.0, 10, 50, 300, and 500 μg ml⁻¹ at37° C. for 15 min. Immediately after incubation, cell suspensions werelysed in Laemmli buffer and heated to 95° C. for 10 min. Lysates wereanalysed by SDS-PAGE and immunoblotting using F1B6 Mab.

Example C.6 Two-Dimensional Gel Electrophoresis

[0163] Two-dimensional gel electrophoresis (2-DGE) was carried outessentially as described by Cordwell et al. ((1997) Electrophoresis18:1393-1398). First dimension immobilized pH gradient (IPG) strips (180mm, linear pH6-11; Amersham Phamracia Biotech, Uppsala, Sweden) wereprepared for focusing by submersion in 2-DGE compatible sample buffer (5M urea, 2 M thiourea, 0.1% carrier ampholytes 3-10, 2% w/v CHAPS, 2% w/vsulfobetaine 3-10, 2 mM tributyl phosphine (TBP; Bio-Rad, Hercules USA))overnight. M. hyopneumoniae whole cell protein (250 μg)) was dilutedwith sample buffer to a volume of 100 μl for application to the anodicend of each IPG strip via an applicator cup. Isoelectric focusing wasperformed with a Multiphor II electrophoresis unit (Amersham PharmaciaBiotech) for 85 kVh at 20° C. IPG strips were detergent exchanged,reduced and alkylated in buffer containing 6 M urea, 2% SDS, 20%glycerol, 5 mM TBP, 2.5% v/v acrylamide monomer, trace amount ofbromophenol blue dye and 375 mM Tris-HCl (pH 8.8) for 20 minutes priorto loading the IPG strip onto the top of an 8-18% T, 2.5% C (piperazinediacrylamide) 20 cm×20 cm polyacrylamide gel. Second-dimensionelectrophoresis was carried out at 4° C. using 3 mA/gel for 2 hours,followed by 20 mA/gel until the bromophenol blue dye had run off the endof the gel. Gels were fixed in 40% methanol, 10% acetic acid for 1 hourand then stained overnight in Sypro Ruby (Molecular Probes, Eugene,Oreg.). Images were acquired using a Molecular Imager Fx (Bio-Rad). Gelswere then double-stained in Coomassie Blue G-250.

Example C.7 Post-Separation Analyses

[0164] Protein spots were excised from gels using a sterile scalpel andplaced in a 96 well tray (Gobom et al. (1999) J. Mass. Spectrom.34:105-116). Gel pieces were washed with 50 mM ammonium bicarbonate/100%acetonitrile (60:40 v/v) and then dried in a Speed Vac (SavantInstruments, Holbrook, N.Y.) for 25 min. Gel pieces were then hydratedin 12 μl of 12 ng μl⁻¹ sequencing grade modified trypsin (Promega,Madison, Wis.) for 1 h at 4° C. Excess trypsin solution was removed andthe gel pieces immersed in 50 mM ammonium bicarbonate and incubatedovernight at 37° C. Eluted peptides were concentrated and desalted usingC₁₈ Zip-Tips™ (Millipore Corp., Bedford, Mass.). The peptides werewashed on a column with 10 μl 5% formic acid. The bound peptides wereeluted from the Zip-Tip™ in matrix solution (10 mg ml⁻¹α-cyano-4-hydroxycinnamic acid [Sigma] in 70% acetonitrile) directlyonto the target plate. Matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry (MALDI-TOF MS) mass spectra wereacquired using either a PerSeptive Biosytems Voyager DE-STR (Framingham,Mass.) or a Micromass TofSpec2E (Micromass, Manchester UK). Bothinstruments were equipped with 337 nm nitrogen lasers. All spectra wereobtained in reflectron/delayed extraction mode, averaging 256 lasershots per sample. Two-point internal calibration of spectra wasperformed based upon internal porcine trypsin autolysis peptides (842.5and 2211.10 [M+H]⁺ ions). A list of monoisotopic peaks corresponding tothe mass of generated tryptic peptides was used to search a modifiedtranslated version of the M. hyopneumoniae genome. Successfulidentifications were based on the number of matching peptide masses andthe percentage sequence coverage afforded by those matches. N-terminalEdman sequencing was performed as previously described (Nouwens et al.(2000) Electrophoresis 21:3797-3809).

Example C.8 Immunoelectron Microscopy

[0165]M. hyopneumoniae strain 232 cells were grown to mid log phase,pelleted by centrifugation and washed with phosphate buffered saline(PBS). The final cell pellets were fixed with 3% glutaraldehyde in 0.1 Msodium cacodylate buffer (pH 7.2) at 4° C. overnight. The pellets werewashed three times with 0.1 M sodium cacodylate buffer, 15 min betweenchanges and post fixed with 1% osmium tetroxide in 0.1 M sodiumcacodylate buffer for 2 h at room temperature. The pellets were thenwashed with distilled water, passed through an acetone series andembedded in Embed 812 and Araldite (Electron Microscopy Sciences, FortWashington, Pa.). Thin sections (80-90 nm) were then washed six timeswith TS buffer, and reacted with F1B6 ascites fluid (diluted 1:50),anti-ΔNP97 ascites fluid (diluted 1:10), anti-NP97 ascites fluid(diluted 1:10), or mouse anti-human fibronectin (diluted 1:25) overnightat 4° C. The grids were washed five times with TS buffer and thenreacted with goat anti-mouse IgG+IgM labeled with 10 nm colloidal goldparticles (EY Laboratories, Inc.) diluted 1:25 for 30 min at roomtemperature. The cells were then washed 5 times with TS buffer, dried,contrasted with osmium vapors for 2 min, and stained with uranylacetate-lead citrate. The sections were examined on a Hitachi 500 at 75kV.

[0166] For tracheal sections, mycoplasma-free pigs were inoculatedintratracheally with M. hyopneumoniae strain 232. At 10 and 21 days,pigs were sacrificed, tracheas were removed and 1 cm blocks of tissuefixed with 1% glutaraldehyde overnight, dehydrated in an acetone series,and embedded as above. Thick (1-2 μm) sections were stained withmethylene blue polychrome and examined by microscopy for regionscontaining ciliated epithelium. Thin sections (80-90 nm) were thenprepared for labeling. The sections were pretreated with ammoniumchloride (1%) for 1 h, 0.05 M glycine in PBS for 15 min, blocked for 30min in 2% fish gelatin+2% bovine serum albumin in TS buffer (10 mM Tris,100 mM NaCl, pH 7.5). Primary antibodies were diluted in TS buffer andreacted with sections for 30 min at room temperature. The sections werewashed six times with TS buffer, and then incubated with goat anti-mouseIgG+IgM labeled with 10 nm gold particles (diluted 1:2) for 15 min atroom temperature. Both primary antibodies and the conjugate were dilutedand centrifuged briefly (12,000×g for 5 min) prior to use. The sectionswere then washed six times with TS buffer, dried, contrasted with osmiumvapors for 2 min, and stained with uranyl acetate-lead citrate. Thesections were examined on a Hitachi 500 at 75 kV.

Example C.9 Fibronectin Binding Assay

[0167] Immunlon 2 (Dynatech Laboratories, Inc.) 96 well plates werecoated with 100 μl of human fibronectin (Sigma, F 0895) at aconcentration of 5 μg ml⁻¹ in 0.1 M sodium carbonate. Plates wereincubated at 4° C. overnight, washed three times with PBS, and blockedwith 1% bovine serum albumin in PBS for 2 hr. The plates were thenincubated with purified recombinant P97 with or without inhibitor at aconcentration of 10 μg ml⁻¹. Inhibitors tested were intact humanfibronectin, 45-kDa proteolytic fragment of fibronectin (Sigma, F 0162),30-kDa proteolytic fragment of fibronectin (Sigma, F 9911) andengineered RGD polymer (Sigma, 5022). They were added to Eppendorf tubeswith purified recombinant P97 (10 μg ml⁻¹) at concentrations of 37.5 μgml⁻¹, 7.5 μg ml⁻¹, and 1.5 μg ml⁻¹ and incubated at 37° C. for 1 hr. Therecombinant P97 plus inhibitor was then transferred to a fibronectincoated plate, which was then incubated at 37° C. for 2 hr. Binding ofP97 to fibronectin was assessed by ELISA with Mab F1B6. Optical densityat 405 nm was indicative of P97 binding to fibronectin-coated wells.Three replicates per treatment were assayed from three differentexperiments. Statistical differences were determined by the GeneralLinear Model with a linear contrast based on pooled variances.

Example C.10 Results of Two-Dimensional Gel Electrophoresis and MassSpectrometry

[0168] Previous studies have demonstrated that the gene product for thecilium adhesin of strain 232 (126-kDa preprotein, 1036 amino acids)undergoes a cleavage event at amino acid 195 to yield what was oncethought to be the “mature” molecule (Hsu et al. (1997) J. Bacteriol.179:1317-1323). During peptide mass mapping studies of J strainproteins, four spots of 22, 28, 66 and 94 kDa (subsequently referred toas P22, P28, P66 and P94, respectively) were identified that representeddifferent fragments of the adhesin. The N-terminal sequences for theseproteins allowed unequivocal alignment with the cilium adhesinpreprotein. P94 of strain J, the homologue of P97 in strain 232, mappedto a region that begins immediately downstream of amino acid 195 untilthe end of the ORF. Two closely spaced proteins at 66 kDa had identicalmass maps and corresponded to a region beginning immediately downstreamof amino acid 195 of the adhesin and ending near the R1 repeat.N-terminal sequence analysis of P66 showed a sequence of ADEKTSS (SEQ IDNO:41) that is identical to that of P94. Immunoblotting results usingMab F1B6 confirmed that P66 contains R1. Thus, the cleavage event mustoccur immediately downstream of the R1 repeat region. These data suggestthat a fragment approximately 28 kDa in size had been removed from theC-terminus in some, but not all of the P94 molecules. This observationwas confirmed when a 28-kDa fragment was identified that mapped to theC-terminus of P94. Also, one and two-dimensional immunoblots of J strainproteins probed with antisera raised against a recombinant 28-kDaprotein containing R2 but not R1 (Wilton et al. (1998) Microbiology144:1931-1943) recognised both P28 and P94 proteins. Previously, it wasshown that antisera raised against a 28-kDa C-terminal recombinantpeptide of the adhesin recognised the mature form of this antigen (93-97kDa) in different strains of M. hyopneumoniae and a 28-kDa fragment onlyin strain J (Wilton et al. (1998) Microbiology 144:1931-1943). Trypticpeptide mass mapping showed that peptides from P22 mapped to the first190 amino acids of the 123-kDa adhesin preprotein. The N-terminalsequence of P22 (SKKSKTF (SEQ ID NO:42)) aligned to amino acids 2-8 inthe N-terminus of the 123 kDa preprotein suggesting that cleavage of thehydrophobic leader peptide (amino acids 8-22) is not necessary fortranslocation of the cilium adhesin across the membrane.

[0169] Comparative peptide mass mapping studies of strain 232 identifiedtwo spots of 70 and 97 kDa, subsequently identified as P70 and P97,respectively. Mass maps representative of P97 corresponded to a regionbeginning immediately downstream of amino acid 195 until the end of theORF and corresponded to the most abundant product of the 232 strainadhesin gene (Zhang et al. (1995) Infect. Immun. 63:1013-1019).Interestingly, mass maps representative of P70 corresponded to a regionbeginning immediately downstream of amino acid 195 and ending near theR1 repeat, a map that was virtually identical to P66 in strain J. Thepresence of six extra copies of the R1 repeat is the most likelyexplanation for the difference in masses between P66 and P70 in strainsJ and 232, respectively. Consistent with these data, immunoblots probedwith antisera raised against a recombinant 28-kDa protein containing R2but not R1 (Wilton et al. (1998) Microbiology 144:1931-1943) recognizedP97 but not P70 or P28. Furthermore, P28 or P22 could not be identifiedon 2D gels of 232 proteins resolved by 2D gel electrophoresis in regionswhere they were identified in strain J. This variation was not due todifferences in sequence since P22 sequences were identical in the twostrains. This was not true for the P28 sequences, however. The predictedmass and pI for P28 from strain 232 was 24.6 kDa and 5.88, respectively,and for P28 from strain J, it was 26.0 kDa and 8.39. It was possiblethat P28 was not found in strain 232 because of the change in pI causinga shift in the gel location of the protein. It was also possible thatadditional cleavage of P22 occurred in strain 232 that did not in strainJ.

[0170] To rule out the possibility that cleavage resulted from aproteolytic activity in the media used for culturing M. hyopneumoniae,purified recombinant P97 was incubated with fresh and spent medium andthen examined for proteolytic cleavage by immunoblot. Because the mediumcontained 20% swine serum, large quantities of swine immunoglobulinswere present in the protein samples causing some background stainingwith the anti-mouse conjugate. It was still clear, however, that neitherfresh nor spent medium contained proteolytic activity capable ofcleaving recombinant P97 after 12 hours of incubation at 37° C. Thus,cleavage of the cilium adhesin was mediated by mycoplasma-encodedactivities and was not due to porcine serum or other medium components.

Example C.11 Trypsin Sensitivity of R1-Containing Cleavage Products

[0171] Immunoblot analyses of strain J and 232 cells digested withdifferent concentrations of trypsin was used to investigate the cellularlocation of R1-containing cleavage fragments. The F1B6 Mab typicallyrecognised proteins with masses of 35, 66, 88, 94, and 123 kDa in strainJ and a similar pattern was observed for strain 232. Exposure of intactM. hyopneumoniae to concentrations of trypsin ranging from 0.1-10 μgml⁻¹ showed a gradual loss of the higher mass proteins. Concentrationsbetween 10 and 50 μg ml⁻¹ resulted in the loss of all the immunoreactiveproteins (except one of 35 kDa) indicating that R1-containing adhesinfragments are surface accessible. The pattern of digestion ofR1-containing adhesin fragments was consistent in repeat experimentsexcept that the 35 kDa fragment was not reliably resistant to trypsin atconcentrations above 10 μg ml⁻¹. Identical blots reacted with antiseraraised to recombinant M. hyopneumoniae lactate dehydrogenase (previouslyshown to reside cytosolically) (Strasser et al. (1991) Infect. Immun.59:1217-1222) and to antisera raised to recombinant fragments ofpyruvate dehydrogenase subunits A and D showed that these proteinsremained detectable with trypsin concentrations up to 500 μg ml⁻¹. Incontrol experiments where lysed cells were exposed to trypsin, lactatedehydrogenase and pyruvate dehydrogenase subunit D were rapidlydegraded.

Example C.12 Results of Immunogold Electron Microscopy

[0172] Transmission electron microscopy studies have shown that high andlow adherent strains of M. hyopneumoniae differ in their outer membranestructure. High adherent clones possessed fibrils on the outer surfacethat appeared to interconnect to adjacent cells; these fibrils wererarely observed in low adherence clones (Young et al. (1994) Isolationand characterization of high and low adherent clones of Mycoplasmahyopneumoniae. In IOM Letters. 10^(th) International Congress of theInternational Organization for Mycoplasmology. Vol. 3 Bordeaux, France,pp. 684-685). Antisera generated against specific regions of the adhesinenabled analysis of cleavage in vivo using immunogold electronmicroscopy. Virulent strain 232 was used in these studies because theseresults would have the most impact on understanding pathogenicmechanisms. R1-specific Mab F1B6 and antisera raised to peptidesTSSQKDPST (ΔNP97 antiserum) (SEQ ID NO:39) and VNQNFKVKFQAL (NP97antiserum) (SEQ ID NO:40) were used in these studies. The Mab F1B6remained associated with the mycoplasma membrane, but not intimatelyassociated with the cell confirming a previous report (Zhang et al.(1995) Infect. Immun. 63:1013-1019) and the trypsin studies above. ΔNP97antiserum showed that this portion of the molecule is located distal tothe membrane in association with extracellular material of unknowncomposition. In some instances, the antibodies seemed to definefibrial-like structures still attached to the mycoplasma cell membrane.NP97 antibodies clustered in aggregates to cytosolic locations,intimately to the membrane surface, and were also observed at sitesdistant from the extracellular surface of the cell membrane.

Example C.13 Fibronectin Binding Results

[0173] Since cleavage of the cilium adhesin occurs at amino acidposition 195 (Hsu et al. (1997) J. Bacteriol. 179:1317-1323), it was notreadily apparent how the remaining adhesin could remain associated withthe cell and direct binding to porcine cilia. Immunogold studies showedthat all cilium binding R1 epitopes remained cell associated in theabsence of the hydrophobic N-terminus sequence, but apparently are notinserted directly into the membrane. This is not surprising since noother region of the protein has sufficient hydrophobicity to directmembrane insertion (Hsu et al. (1997) J. Bacteriol. 179:1317-1323). Thepossibility that other proteins may play a role in bridgingR1-containing protein fragments of the cilium adhesin to the membranethrough protein-protein interactions was examined. Analysis of thepredicted protein sequence of the 123 kDa adhesin preprotein with thecomputer program COILS (http://www.ch.embnet.org) revealed that theprotein contained three coiled coil domains. One of these residedbetween amino acids 180-195 in P22 (14-, 21- and 28-amino acid windowsettings) and two were located in P97 between amino acids 367-387(window setting 14) and 780-805 (window setting 14 and 21). Thesedomains are known to mediate protein-protein interactions. In addition,it was thought that the R1 and R2 domains might also play a role ininteractions with other proteins. One obvious protein to test wasfibronectin, a protein found in abundance throughout the host and shownto participate in other bacterial-host interactions (Probert et al.(2001) Infect. Immun. 69:4129-4133; Talay et al. (2000) Cell Microbiol.2:521-535; Rocha and Fischetti (1999) Infect. Immun. 67:2720-2728; andSchorey et al. (1996) Mol. Microbiol. 21:321-329).

[0174] Ligand blotting studies confirmed that recombinant P97 boundporcine fibronectin. Other fibronectin binding proteins were alsoidentified in lysates of M. hyopneumoniae low (lane 1) and high (lane 2)adherent variants of strain 232 and in strain J (lane 3). The low andhigh adherent strains of 232 differed by the absence of afibronectin-binding band at approximately 50 kDa, which was also presentin strain J.

[0175] Fibronectin binding assays with human fibronectin and purifiedrecombinant cilium adhesin were also performed. Maximum inhibitionoccurred with the engineered RGD domain at all three concentrationstested (p<0.001). Inhibition also occurred with intact fibronectin(p<0.001) as expected. Interestingly, the 45-kDa purified fragment offibronectin enhanced binding at the highest concentration tested.

[0176] To investigate the role(s) fibronectin might play in the bindingof M. hyopneumoniae to porcine respiratory epithelial cells,anti-fibronectin antibodies were applied to lung sections showing M.hyopneumoniae strain 232 in close association with respiratoryepithelial cilia. Gold particles were localised in regions where M.hyopneumoniae cells were intimately associated with cilia, on thesurface of cilia and on the surface of M. hyopneumoniae cells.

[0177] D. Detection of Infection and Immunogenic Compositions

Example D.1 Detection of M. hyopneumoniae Infection in Swine

[0178] The polypeptides displaying M. hyopneumoniae antigenicity of thisinvention may be used in methods and kits designed to detect thepresence of M. hyopneumoniae infection in swine herds and therefore torecognize swine in a herd which have been infected by this bacteria. Forexample, the antigens produced by hosts transformed by recombinantnucleic acid molecules of this invention, or antibodies raised againstthem, can be used in RIA or ELISA for these purposes. In one type ofradioimmunoassay, antibody against one or more of the antigens of thisinvention, raised in a laboratory animal (e.g., rabbits), is attached toa solid phase, for example, the inside of a test tube. Antigen is thenadded to the tube to bind with the antibody.

[0179] A sample of swine serum, taken from 1 of each 10 to 20 swine perherd, together with a known amount of antigen antibody labeled with aradioactive isotope, such as radioactive iodine, is then added to thetube coated with the antigen-antibody complex. Any antigen (a marker forM. hyopneumoniae infection) antibody in the swine serum will competewith the labeled antibody for the free binding sites on antigen-antibodycomplex. Once the serum has been allowed to interact, the excess liquidis removed, the test tube washed, and the amount of radioactivitymeasured. A positive result, i.e., that the tested swine's serumcontains M. hyopneumoniae antibody, is indicated by a low radioactivecount.

[0180] In one type of ELISA test, a microtiter plate is coated with oneor more antigens of this invention and to this is added a sample ofswine serum, again, from 1 in every 10 or 20 swine in a herd. After aperiod of incubation permitting interaction of any antibody present inthe serum with the antigen, the plate is washed and a preparation ofantigen antibodies, raised in a laboratory animal and linked to anenzyme label, is added, incubated to allow reaction to take place, andthe plate is then rewashed. Thereafter, enzyme substrate is added to themicrotiter plate and incubated for a period of time to allow the enzymeto work on the substrate, and adsorbance of the final preparation ismeasured. A large change in adsorbance indicates a positive result,i.e., the tested swine serum had antibodies to M. hyopneumoniae and wasinfected with that bacteria.

Example D.2 Immunogenic Compositions

[0181] Standard methods known to those skilled in the art may be used inpreparing immunogenic compositions of polypeptides and nucleic acids ofthe present invention for administration to swine. For example, thepolypeptide of choice may be dissolved in sterile saline solution. Forlong-term storage, the polypeptide may be lyophilized and thenreconstituted with sterile saline solution shortly beforeadministration. Prior to lyophilization, preservatives and otherstandard additives such as those to provide bulk, e.g., glycine orsodium chloride, may be added. A compatible adjuvant may also beadministered with the composition.

[0182] In addition, compositions can be prepared using antibodies raisedagainst the polypeptides of this invention in laboratory animals, suchas rabbits. This “passive” vaccine can then be administered to swine toprotect them from M. hyopneumoniae infection. Direct incorporation ofnucleic acid sequences into host cells may also be used to introduce thesequences into animal cells for expression of antigen in vivo.

[0183] The above description, drawings and examples are onlyillustrative of preferred embodiments that achieve the objects, featuresand advantages of the present invention. It is not intended that thepresent invention be limited to the illustrated embodiments. Anymodification of the present invention that comes within the spirit andscope of the following claims should be considered part of the presentinvention.

Other Embodiments

[0184] It is to be understood that while the invention has beendescribed in conjunction with the detailed description thereof, theforegoing description is intended to illustrate and not limit the scopeof the invention, which is defined by the scope of the appended claims.Other aspects, advantages, and modifications are within the scope of thefollowing claims.

1 42 1 3029 DNA Mycoplasma hyopneumoniae 1 atgaaaaaaa tacctaattttaaaggattt tttaataaac cagcaaaaat tgtaactagc 60 attttgcttc taagtggtattataactatt tcaactgcaa ttcctttagg tatttggtca 120 tataatcgcg cttattatcaaaaattaaat gaaaaatcac aaaatttaag tattagtcaa 180 actgaaaatc cctttgaaaataatcttgga aaattctttg ataatttatt cattagtaat 240 caattcaaag aattatcagctagtacagca tttgaattag caaaaagcaa gatttataat 300 cttgaccttt taacgttaattaatcttgat aaactatacc aaaaaaatta ccaaattagt 360 tatgatctaa gtaatgcaacagcaagtgga actgcaatta aaaatattgt attttttata 420 agaactagcg atcaacggcaaattttttca aaagcagttg aaattaaagg tttttctgat 480 aaaaatattg aaaaaaatcttgctaaattt gaaattgaag aaaaaaaatc atcaatttca 540 attaaaccgc aaaattttttaagttttgct gagtttagca aggaattaca aaatcaattt 600 attaaaacta gcaaaacccaaaaacaaaca tttattgctt ttgaagaggc gcttattcaa 660 cttggaggtt cgtataatttagttaacagt ctcggcttac caacttttat tcataaaggg 720 caaattttag aaccaaaaatttttgataat aatcttaatt ttacaaacca agggaataaa 780 aattacctta attttatcttcacaaatgaa ggaaaaaaaa cagaaattcc cttagaaatt 840 aacggaataa cccctgatttagagattaaa aatgaaataa ttaagtgaat aaaagcggaa 900 ctagaagaaa aaatcaagctcaaggaaagt attcaagctg aattaattag ggaaaattta 960 tcacttgcaa aatcattttatgttgataaa aataataatc ctttgatatc aacaacaaaa 1020 aattttgaaa acttatttgattatgtacaa agcgagcatc taattaatac taataaaata 1080 aaaaattata tcacaaacataaattttaaa atcaaaaaaa atagtgaaat acctgcttta 1140 gaacttaata atttgctaaaagatgataaa attcggcttg aataaatgtt gatatctcaa 1200 agtgagtcca acaaaaactaattaaaattt taaattttaa gtttgattgg gacctaaaac 1260 cagacctgaa tcagtatgccaggatttttg cacaaaatct acccgagcca aaatctgagg 1320 tattcttact aaaaaaagatgaaaattcag cagcgtgaac tagtaaaaaa ctagtaaata 1380 taataaataa aattaaggaatttaacaatg aattagaccc agaaaatcct gatataaagc 1440 tagttagcca actttatttacttgattttg gcaaaattgg tgatgaaatt gctatagaaa 1500 attataaaag agaattaataataactgcta aaatccttaa aaatcaacta gttaaagtcc 1560 aagaatttag tgatgatcaggttaataaag cacaaaacaa tgaaaaaagt ttaggaaaag 1620 caatttgtaa agtgcttaatattcagcgta atttaataaa tgatgatata agctctgatt 1680 ttatccttga taataaggaaggtgatttta ctatcgaatt tagtctaatt tcaaataaaa 1740 ataagcaaaa attagccacaagaaagatta aaatttcaaa tattgtcagt tctgaaatga 1800 gcgcttttga tgatgcagctaaattttatc caactttttt tcttgatggc aagtcatctt 1860 tttcaaaatc agacaataaaaaaggctatg aaattataga tttatctgat aataatattc 1920 attttgagga tgatttagatagtaaaaatc aactaactca agaaggtttt aaactaacaa 1980 atccgattaa atttcagcaaaaccaatcaa aaacaaaaga aaatattgcc agaacagtca 2040 atataagtag cccaagtttcaaatcagcac cattttcacg gcttgattca gggctaattt 2100 atttagcatt taaaccaaaaaatatcaatg actataaaaa acattaccta cttgcagact 2160 cagatggaaa cggtctttttattcaaaaga ttaaaaattt taaatttata aataaaaata 2220 ccacaatcca agggattgcaggactaaaaa ctgaaaaaac tacgcaaaat tcggatatta 2280 cctttatcaa acccgaaaatttagaccaaa aaaaaaaaga tgaaaaaaaa caaaaacaag 2340 ttgatggtta ttttatcggacttgacttta aacagataaa aaattttaaa tcatttcagt 2400 catatttgta ccagaacaaaaaaagctatt attccttagc taatttattc ccacctgaat 2460 taattgataa gcaagcagtaattcttgggc ctaattcctg aaagccaata aaatatttta 2520 gcgctgaaat aaatcaaaatttagacaatc tagccatagt tgaacttgca aatcgaattg 2580 gcgaaaatcg tttttatcgccaggaactaa gaaattctag tcctttttca cttgaaaaaa 2640 gtaaagaaat aatcgaagaagaccaagata ttgtccttga aattatcaaa actccgtgat 2700 cagttgaaat tagtgctttttcatcatcaa attatcaact aaattcaaaa acatcactta 2760 atttaaatgg aaaaactatctataatatta accctgtaag tcaaaaatgg tcaccatttc 2820 cgaattatct aaatcttgactgggcccaaa ttgggccaaa tccaaaaaaa acaacggata 2880 aaaatggttc taacaacgaaaaaattaaca aaaatagcag cataaattta aaaggaatag 2940 cagtttataa cgatccagaattaacaacaa agacaagaaa ttttgcccgc gatcaaataa 3000 gaaacgcctt tattaaagcatatataaaa 3029 2 1009 PRT Mycoplasma hyopneumoniae 2 Met Lys Lys Ile ProAsn Phe Lys Gly Phe Phe Asn Lys Pro Ala Lys 1 5 10 15 Ile Val Thr SerIle Leu Leu Leu Ser Gly Ile Ile Thr Ile Ser Thr 20 25 30 Ala Ile Pro LeuGly Ile Trp Ser Tyr Asn Arg Ala Tyr Tyr Gln Lys 35 40 45 Leu Asn Glu LysSer Gln Asn Leu Ser Ile Ser Gln Thr Glu Asn Pro 50 55 60 Phe Glu Asn AsnLeu Gly Lys Phe Phe Asp Asn Leu Phe Ile Ser Asn 65 70 75 80 Gln Phe LysGlu Leu Ser Ala Ser Thr Ala Phe Glu Leu Ala Lys Ser 85 90 95 Lys Ile TyrAsn Leu Asp Leu Leu Thr Leu Ile Asn Leu Asp Lys Leu 100 105 110 Tyr GlnLys Asn Tyr Gln Ile Ser Tyr Asp Leu Ser Asn Ala Thr Ala 115 120 125 SerGly Thr Ala Ile Lys Asn Ile Val Phe Phe Ile Arg Thr Ser Asp 130 135 140Gln Arg Gln Ile Phe Ser Lys Ala Val Glu Ile Lys Gly Phe Ser Asp 145 150155 160 Lys Asn Ile Glu Lys Asn Leu Ala Lys Phe Glu Ile Asp Glu Lys Lys165 170 175 Ser Ser Ile Ser Ile Lys Pro Gln Asn Phe Leu Ser Phe Ala GluPhe 180 185 190 Ser Lys Glu Leu Gln Asn Gln Phe Ile Lys Thr Ser Lys ThrGln Lys 195 200 205 Gln Thr Phe Ile Ala Phe Glu Glu Ala Leu Ile Gln LeuGly Gly Ser 210 215 220 Tyr Asn Leu Val Asn Ser Leu Gly Leu Pro Thr PheIle His Lys Gly 225 230 235 240 Gln Ile Leu Glu Pro Lys Ile Phe Asp AsnAsn Leu Asn Phe Thr Asn 245 250 255 Gln Gly Asn Lys Asn Tyr Leu Asn PheIle Phe Thr Asn Glu Gly Lys 260 265 270 Lys Thr Glu Ile Pro Leu Glu IleAsn Gly Ile Thr Pro Asp Leu Glu 275 280 285 Ile Lys Asn Glu Ile Ile LysTrp Ile Lys Ala Glu Leu Glu Glu Lys 290 295 300 Ile Lys Leu Lys Glu SerIle Gln Ala Glu Leu Ile Arg Glu Asn Leu 305 310 315 320 Ser Leu Ala LysSer Phe Tyr Val Asp Lys Asn Asn Asn Pro Leu Ile 325 330 335 Ser Thr ThrLys Asn Phe Glu Asn Leu Phe Asp Tyr Val Gln Ser Glu 340 345 350 His LeuIle Asn Thr Asn Lys Ile Lys Asn Tyr Ile Thr Asn Ile Asn 355 360 365 PheLys Ile Lys Lys Asn Ser Glu Ile Pro Ala Leu Glu Leu Asn Asn 370 375 380Leu Leu Lys Asp Asp Lys Ile Arg Leu Glu Ile Asn Val Asp Ile Ser 385 390395 400 Lys Trp Val Gln Gln Lys Leu Ile Lys Ile Leu Asn Phe Lys Phe Asp405 410 415 Trp Asp Leu Lys Pro Asp Leu Asn Gln Tyr Ala Arg Ile Phe AlaGln 420 425 430 Asn Leu Pro Glu Pro Lys Ser Glu Val Phe Leu Leu Lys LysAsp Glu 435 440 445 Asn Ser Ala Ala Trp Thr Ser Lys Lys Leu Val Asn IleIle Asn Lys 450 455 460 Ile Lys Glu Phe Asn Asn Glu Leu Asp Pro Glu AsnPro Asp Ile Lys 465 470 475 480 Leu Val Ser Gln Leu Tyr Leu Leu Asp PheGly Lys Ile Gly Asp Glu 485 490 495 Ile Ala Ile Glu Asn Tyr Lys Arg GluLeu Ile Ile Thr Ala Lys Ile 500 505 510 Leu Lys Asn Gln Leu Val Lys ValGln Glu Phe Ser Asp Asp Gln Val 515 520 525 Asn Lys Ala Gln Asn Asn GluLys Ser Leu Gly Lys Ala Ile Trp Lys 530 535 540 Val Leu Asn Ile Gln ArgAsn Leu Ile Asn Asp Asp Ile Ser Ser Asp 545 550 555 560 Phe Ile Leu AspAsn Lys Glu Gly Asp Phe Thr Ile Glu Phe Ser Leu 565 570 575 Ile Ser AsnLys Asn Lys Gln Lys Leu Ala Thr Arg Lys Ile Lys Ile 580 585 590 Ser AsnIle Val Ser Ser Glu Met Ser Ala Phe Asp Asp Ala Ala Lys 595 600 605 PheTyr Pro Thr Phe Phe Leu Asp Gly Lys Ser Ser Phe Ser Lys Ser 610 615 620Asp Asn Lys Lys Gly Tyr Glu Ile Ile Asp Leu Ser Asp Asn Asn Ile 625 630635 640 His Phe Glu Asp Asp Leu Asp Ser Lys Asn Gln Leu Thr Gln Glu Gly645 650 655 Phe Lys Leu Thr Asn Pro Ile Lys Phe Gln Gln Asn Gln Ser LysThr 660 665 670 Lys Glu Asn Ile Ala Arg Thr Val Asn Ile Ser Ser Pro SerPhe Lys 675 680 685 Ser Ala Pro Phe Ser Arg Leu Asp Ser Gly Leu Ile TyrLeu Ala Phe 690 695 700 Lys Pro Lys Asn Ile Asn Asp Tyr Lys Lys His TyrLeu Leu Ala Asp 705 710 715 720 Ser Asp Gly Asn Gly Leu Phe Ile Gln LysIle Lys Asn Phe Lys Phe 725 730 735 Ile Asn Lys Asn Thr Thr Ile Gln GlyIle Ala Gly Leu Lys Thr Glu 740 745 750 Lys Thr Thr Gln Asn Ser Asp IleThr Phe Ile Lys Pro Glu Asn Leu 755 760 765 Asp Gln Lys Asn Lys Asp GluThr Gln Gln Lys Gln Val Asp Gly Tyr 770 775 780 Phe Ile Gly Leu Asp PheLys Gln Ile Lys Asn Phe Lys Ser Phe Gln 785 790 795 800 Ser Tyr Leu TyrGln Asn Lys Lys Ser Leu Tyr Ser Leu Ala Asn Leu 805 810 815 Phe Pro ProGlu Leu Ile Asp Lys Gln Ala Val Ile Leu Gly Pro Asn 820 825 830 Ser TrpLys Pro Ile Lys Asn Phe Ser Ala Glu Ile Asn Gln Asn Leu 835 840 845 AspAsn Leu Ala Ile Val Glu Leu Ala Asn Arg Ile Gly Glu Asn Arg 850 855 860Phe Tyr Arg Gln Glu Leu Arg Asn Ser Ser Pro Phe Ser Leu Glu Lys 865 870875 880 Ser Lys Glu Ile Ile Glu Glu Asp Gln Asp Ile Val Leu Glu Ile Ile885 890 895 Lys Thr Pro Trp Ser Val Glu Ile Ser Ala Phe Ser Ser Ser AsnTyr 900 905 910 Gln Leu Asn Ser Lys Thr Ser Leu Asn Leu Asn Gly Lys ThrIle Tyr 915 920 925 Asn Ile Asn Pro Val Ser Gln Lys Trp Ser Pro Phe ProAsn Tyr Leu 930 935 940 Asn Leu Asp Trp Ala Gln Ile Gly Pro Asn Pro LysLys Thr Thr Asp 945 950 955 960 Lys Asn Gly Ser Asn Asn Glu Lys Ile AsnLys Asn Ser Ser Ile Ile 965 970 975 Leu Lys Gly Ile Ala Val Tyr Asn AspPro Glu Leu Thr Thr Lys Thr 980 985 990 Arg Asn Phe Ala Arg Asp Gln IleArg Asn Ala Phe Ile Lys Ala Tyr 995 1000 1005 Ile 3 3096 DNA Mycoplasmahyopneumoniae 3 atgcaggcta atttgattgg cagatttatc aaaaataaaa aagcaattttggtactagct 60 tcaacttttg ctgggttaat tttatttact acttctgtcg gaattagtttaacaattaaa 120 tataatggtt ctcacccgcg ggcaaaagtt aatgaatttg cacaaaaaattagttttgtt 180 agttttaaac ctgagcaaat tagtaaaaat agtaatttct gaaaaataaaagaaaaattg 240 ttttccggtg atcagcttaa aaaagaaata aatcttgaag agtatctccaattttatatt 300 tttgataaaa attctaatga tttggttaaa ttctcaaaag attcaaatcctttttctatt 360 gaatttgaat ttagtgattt aaaatttgat gatttaaacc aaaattttaatcttaaattt 420 cgtgttaggc aaaaacaaaa aaataatcaa tatgcatatt cggattttttcagccaacca 480 attacatttt atgaatcaaa taaattttta aaagcagatt ttaactttgttcttcaaaaa 540 atgtttcgcc aaattaatga aaatatttta aatataggta attttaccacaaatttttct 600 gatcaaacta gtaaaaaaaa attaaaaaag ttatacagag caattgattttgcgcaagaa 660 gttaataaaa ttgaaaatcc aaacgaggtt gaggtcaaaa taaatgaaattttccctgaa 720 ttatctaact tgattttaca agcacgcgaa tcgaaagata ataaaattggaaaaacagaa 780 aatccgattt ttagtcttaa atttataaaa aataaaacta ataatcaatttgtaaatcta 840 caagataata tcccaactat gtatcttgag gcaaaattaa ctgatcaagccgcaaaaatg 900 ttaggtgata ttggtcaaaa ctttagcgaa aaaatctttg aaattagatttgaaactaat 960 gataaaaaat cattattttt caatgttgag aatttttttc aaaatattaaactaaaacca 1020 ctaaaattta acactgaaga aaaagacgga aaattaataa taactaaactgaatcctttt 1080 gacatatttt caaaaattaa atccggaatt ttatctgcca atactaaccaaaattacata 1140 aaaggggtta ttaattcttt attagaagag gatttagctc tagattttgggccgacttca 1200 aaactaattc cacaaaatca aaacggaatt agttttgaaa ttatccaacaaaatgctaaa 1260 ttaaaaaatg aaaatgataa ttatataatt gaaattccct ataaaattttccttagagaa 1320 tccttattta aacctggttc acaaaaaatt atctatgaaa aagagttgtttttaagtatt 1380 ggcggctttg gtatatcaaa taaaaatggt caaaatctaa taattccaggaagccagaaa 1440 gctttaattt atcggagaaa ttcacttttt aatgatgagg aaagtcctgaaaataaattt 1500 atttcaactt ttggtcaacc ggtcatttcg aataatccct taaaaaaagaagaaattgat 1560 aatttattat tgcaacaaga ttataaaggt ttagaaagac agctaaattcattatcacgg 1620 tataatttta attttgataa ttttgaggcc aaagttcggg cttgatctggtaagacatac 1680 ttacctagtt taacagaaat tgcaaatttt cgattaaatc aacaaaaaattgatataaat 1740 tcacaaaatc aagagcaaaa aattgaacta aaaacactac attcacaaagtttttttata 1800 aatccttcgg atgtaacagc tttttttgct gatttaattc agaaaaaaccaagccaaata 1860 gcaaatagtt ttttcttaat tgcaaaggct tttggacttt taaatcaaaatcggactgct 1920 tcgcaaattt ttaataacct ggctggagaa aatatctttg aagctagttcaaaaattgat 1980 tttgataata aaactacaaa tattttaagt tttaataatc atttcgctgatttttataat 2040 caagggtttt tttcatccct ttttcttcca aaatcaataa aagataaattcaataatcta 2100 aaaagcaagt caatttctga tgtaattagt attttagaag accaagaactttttaaagaa 2160 acagctagaa aatttacaag acaacaaatt gaggaaaacc taaaatcaagtgttaaattc 2220 acaacattgg ccgaccttct tttagctttt tattataagg ctagtcaacttgataatttt 2280 ttagggtgaa caaaattaga taccaattta gattatcaaa ttgtgtttcaaaaagaaaat 2340 gaaatttcaa aagctcgtta tgattctgaa attcagaagc taaaaaaacccgaattaaat 2400 tctttagaaa aacaggaaaa cttaaataaa aattctgaaa ttcaaccagaatctaaaaat 2460 ttagactctg ataataacat aaaaaaatca ataaatggaa atttagaaaaagataatact 2520 tataatgcca atgttgataa tgaatatcta acattaaatt tttactatattattggtgat 2580 tctagtcaga aaaaattttt ctttcaaagc ccaattcaaa aaattttaataaatttctca 2640 actcaaaaaa ttgatgaaaa ttctaaaata caagaaaaat tcgataaggtagttgaaagt 2700 gttccggctg atttgttaaa ttatagtgtc agtgaagaaa attttaaaaaaattaaggaa 2760 aaattaacaa ataagcattc acctgaacca aaaaataatg acaataataacgatttagat 2820 ttatatttta aagaaacttc cataaatatt gataaaatta gttcttattttaaagaacaa 2880 tttcccaaag aggagacaaa atttttactt gaaccaagtt ttgaaaactcactaaatacg 2940 gataaactaa cctttttaat aagtttttat cttaataaga aggataaaaatcccaaagat 3000 ttaaaagctg ataataaaaa tgatgaaaat agcccgataa atccaattattgcaaggcag 3060 aaattaaaaa ttataataac aaaaaattct aaaaat 3096 4 1032 PRTMycoplasma hyopneumoniae 4 Met Gln Ala Asn Leu Ile Gly Arg Phe Ile LysAsn Lys Lys Ala Ile 1 5 10 15 Leu Val Leu Ala Ser Thr Phe Ala Gly LeuIle Leu Phe Thr Thr Ser 20 25 30 Val Gly Ile Ser Leu Thr Ile Lys Tyr AsnGly Ser His Pro Arg Ala 35 40 45 Lys Val Asn Glu Phe Ala Gln Lys Ile SerPhe Val Ser Phe Lys Pro 50 55 60 Glu Gln Ile Ser Lys Asn Ser Asn Phe TrpLys Ile Lys Glu Lys Leu 65 70 75 80 Phe Ser Gly Asp Gln Leu Lys Lys GluIle Asn Leu Glu Glu Tyr Leu 85 90 95 Gln Phe Tyr Ile Phe Asp Lys Asn SerAsn Asp Leu Val Lys Phe Ser 100 105 110 Lys Asp Ser Asn Pro Phe Ser IleGlu Phe Glu Phe Ser Asp Leu Lys 115 120 125 Phe Asp Asp Leu Asn Gln AsnPhe Asn Leu Lys Phe Arg Val Arg Gln 130 135 140 Lys Gln Lys Asn Asn GlnTyr Ala Tyr Ser Asp Phe Phe Ser Gln Pro 145 150 155 160 Ile Thr Phe TyrGlu Ser Asn Lys Phe Leu Lys Ala Asp Phe Asn Phe 165 170 175 Val Leu GlnLys Met Phe Arg Gln Ile Asn Glu Asn Ile Leu Asn Ile 180 185 190 Gly AsnPhe Thr Thr Asn Phe Ser Asp Gln Thr Ser Lys Lys Lys Leu 195 200 205 LysLys Leu Tyr Arg Ala Ile Asp Phe Ala Gln Glu Val Asn Lys Ile 210 215 220Glu Asn Pro Asn Glu Val Glu Val Lys Ile Asn Glu Ile Phe Pro Glu 225 230235 240 Leu Ser Asn Leu Ile Leu Gln Ala Arg Glu Ser Lys Asp Asn Lys Ile245 250 255 Gly Lys Thr Glu Asn Pro Ile Phe Ser Leu Lys Phe Ile Lys AsnLys 260 265 270 Thr Asn Asn Gln Phe Val Asn Leu Gln Asp Asn Ile Pro ThrMet Tyr 275 280 285 Leu Glu Ala Lys Leu Thr Asp Gln Ala Ala Lys Met LeuGly Asp Ile 290 295 300 Gly Gln Asn Phe Ser Glu Lys Ile Phe Glu Ile ArgPhe Glu Thr Asn 305 310 315 320 Asp Lys Lys Ser Leu Phe Phe Asn Val GluAsn Phe Phe Gln Asn Ile 325 330 335 Lys Leu Lys Pro Leu Lys Phe Asn ThrGlu Glu Lys Asp Gly Lys Leu 340 345 350 Ile Ile Thr Lys Leu Asn Pro PheAsp Ile Phe Ser Lys Ile Lys Ser 355 360 365 Gly Ile Leu Ser Ala Asn ThrAsn Gln Asn Tyr Ile Lys Gly Val Ile 370 375 380 Asn Ser Leu Leu Glu GluAsp Leu Ala Leu Asp Phe Gly Pro Thr Ser 385 390 395 400 Lys Leu Ile ProGln Asn Gln Asn Gly Ile Ser Phe Glu Ile Ile Gln 405 410 415 Gln Asn AlaLys Leu Lys Asn Glu Asn Asp Asn Tyr Ile Ile Glu Ile 420 425 430 Pro TyrLys Ile Phe Leu Arg Glu Ser Leu Phe Lys Pro Gly Ser Gln 435 440 445 LysIle Ile Tyr Glu Lys Glu Leu Phe Leu Ser Ile Gly Gly Phe Gly 450 455 460Ile Ser Asn Lys Asn Gly Gln Asn Leu Ile Ile Pro Gly Ser Gln Lys 465 470475 480 Ala Leu Ile Tyr Arg Arg Asn Ser Leu Phe Asn Asp Glu Glu Ser Pro485 490 495 Glu Asn Lys Phe Ile Ser Thr Phe Gly Gln Pro Val Ile Ser AsnAsn 500 505 510 Pro Leu Lys Lys Glu Glu Ile Asp Asn Leu Leu Leu Gln GlnAsp Tyr 515 520 525 Lys Gly Leu Glu Arg Gln Leu Asn Ser Leu Ser Arg TyrAsn Phe Asn 530 535 540 Phe Asp Asn Phe Glu Ala Lys Val Arg Ala Trp SerGly Lys Thr Tyr 545 550 555 560 Leu Pro Ser Leu Thr Glu Ile Ala Asn PheArg Leu Asn Gln Gln Lys 565 570 575 Ile Asp Ile Asn Ser Gln Asn Gln GluGln Lys Ile Glu Leu Lys Thr 580 585 590 Leu His Ser Gln Ser Phe Phe IleAsn Pro Ser Asp Val Thr Ala Phe 595 600 605 Phe Ala Asp Leu Ile Gln LysLys Pro Ser Gln Ile Ala Asn Ser Phe 610 615 620 Phe Leu Ile Ala Lys AlaPhe Gly Leu Leu Asn Gln Asn Arg Thr Ala 625 630 635 640 Ser Gln Ile PheAsn Asn Leu Ala Gly Glu Asn Ile Phe Glu Ala Ser 645 650 655 Ser Lys IleAsp Phe Asp Asn Lys Thr Thr Asn Ile Leu Ser Phe Asn 660 665 670 Asn HisPhe Ala Asp Phe Tyr Asn Gln Gly Phe Phe Ser Ser Leu Phe 675 680 685 LeuPro Lys Ser Ile Lys Asp Lys Phe Asn Asn Leu Lys Ser Lys Ser 690 695 700Ile Ser Asp Val Ile Ser Ile Leu Glu Asp Gln Glu Leu Phe Lys Glu 705 710715 720 Thr Ala Arg Lys Phe Thr Arg Gln Gln Ile Glu Glu Asn Leu Lys Ser725 730 735 Ser Val Lys Phe Thr Thr Leu Ala Asp Leu Leu Leu Ala Phe TyrTyr 740 745 750 Lys Ala Ser Gln Leu Asp Asn Phe Leu Gly Trp Thr Lys LeuAsp Thr 755 760 765 Asn Leu Asp Tyr Gln Ile Val Phe Gln Lys Glu Asn GluIle Ser Lys 770 775 780 Ala Arg Tyr Asp Ser Glu Ile Gln Lys Leu Lys LysPro Glu Leu Asn 785 790 795 800 Ser Leu Glu Lys Gln Glu Asn Leu Asn LysAsn Ser Glu Ile Gln Pro 805 810 815 Glu Ser Lys Asn Leu Asp Ser Asp AsnAsn Ile Lys Lys Ser Ile Asn 820 825 830 Gly Asn Leu Glu Lys Asp Asn ThrTyr Asn Ala Asn Val Asp Asn Glu 835 840 845 Tyr Leu Thr Leu Asn Phe TyrTyr Ile Ile Gly Asp Ser Ser Gln Lys 850 855 860 Lys Phe Phe Phe Gln SerPro Ile Gln Lys Ile Leu Ile Asn Phe Ser 865 870 875 880 Thr Gln Lys IleAsp Glu Asn Ser Lys Ile Gln Glu Lys Phe Asp Lys 885 890 895 Val Val GluSer Val Pro Ala Asp Leu Leu Asn Tyr Ser Val Ser Glu 900 905 910 Glu AsnPhe Lys Lys Ile Lys Glu Lys Leu Thr Asn Lys His Ser Pro 915 920 925 GluPro Lys Asn Asn Asp Asn Asn Asn Asp Leu Asp Leu Tyr Phe Lys 930 935 940Glu Thr Ser Ile Asn Ile Asp Lys Ile Ser Ser Tyr Phe Lys Glu Gln 945 950955 960 Phe Pro Lys Glu Glu Thr Lys Phe Leu Leu Glu Pro Ser Phe Glu Asn965 970 975 Ser Leu Asn Thr Asp Lys Leu Thr Phe Leu Ile Ser Phe Tyr LeuAsn 980 985 990 Lys Lys Asp Lys Asn Pro Lys Asp Leu Lys Ala Asp Asn LysAsn Asp 995 1000 1005 Glu Asn Ser Pro Ile Asn Pro Ile Ile Ala Arg GlnLys Leu Lys Ile 1010 1015 1020 Ile Ile Thr Lys Asn Ser Lys Asn 1025 10305 3582 DNA Mycoplasma hyopneumoniae 5 atgaaccaat ttgacgaaaa agagaaacaacataataaag caaaagcaat tctttcaacc 60 ggattttcgg ttacatcaat tgcaactacagttgtagcag tcccaattgg actaacaatt 120 tttgagaaat catttagttc ccaagtttcaggaggagtcg ataagaacaa agttgtggat 180 ttaaaatcag attcagatca aatcttctcagaagaagatt ttataagagc agttgagaat 240 cttaaacttt ttgataaata tagacatctaacagcaagaa tggcattagg tcttgccagg 300 gaagcagcta atgcctttaa ctttttagatacttacgact acaccccaat tacaaagcat 360 tcatttaaga tttctttgga tatttccgatgcctttgcgg ctaataaaga agtaaaagcg 420 gtagtagtta gtgcatattc ccaaaaatatcaagttacct attcaagact aacttctcta 480 aaaggttgaa aagaagaaga tgattttggcgatgatatta tagattatca aattaatcaa 540 gagctttcag gtctatcact ttcttccctagcccctgaaa gcgcgcatct tttagcctca 600 gaaatggctt ttcggcttga taatgactttcaagttgcat ataaaaaaac aggatcaaga 660 gccgaggctt ttcgccaggc cttgataaaaaattatcttg gttataactt agttaaccgc 720 caaggtttgc ccactatgct ccaaaagggttatgtgctag cccccaaaac aattgaaaat 780 aaaaatgcaa gcgaagaaaa attagtaaatataaatgaaa atgaccgtgc aagggttaat 840 aaactacaaa aagtagaaaa tctagcctttaaaaacttaa gcgatccaaa tggaacgctt 900 tctattactt ttgaactctg agatccaaatggtaaattag tatccgaata cgattttaaa 960 attaagggaa tcaaaaaact tgattttgatcttaaaaaac aagaggaaaa agtacttcaa 1020 aaggtaactg aatttgttga gattaaaccttatgttcaat taggtttaat ccgtgataat 1080 ttatcattgt ctgaaattat ctataaaagtgataataatc cggagtatct taggaaaata 1140 ttagctaaac taaaagaaca caataacaacaaaagggtgg ataataatac atccactact 1200 aaatttcaag aagaggatct taaaaacgaaccaaattcta atggatcaga acaagattct 1260 ttcgagaaag caaaggaaaa tttccttagtttttttgatc taagatcgag actaattcca 1320 attcccgatc ttcctttata ttatcttaaagttaattcaa ttaattttga tagaaatatt 1380 gaagaaaatg aaaaagaaaa attattaaaaaatgaacaag tagtactcaa agtagatttt 1440 agtcttaaaa aagttgttag cgatattagagccccttatt tagtttctag tcaggttaga 1500 tcaaattatc ccccggtttt gaaagcttcgctagcaaaaa taggtaaggg gtcaaattca 1560 aaagttgtcc ttttagatct tggaaatttatcttcaagat ttaaagttca acttgattat 1620 agtgcaaaac aaagagaaat aattaatactttattaaagg aaaatccaga aagagaaaaa 1680 gaattacaag ctaaaattga aagtaagacgtttagtccaa tagatcttaa caatgatgat 1740 ctattagcaa tcgaatttca atatgaggataaccctgaag gagattgaat aactttaggg 1800 agaatggaaa agttagtcaa agaggttatccaatataaaa aggaaggtaa aaccttccta 1860 gatgatgaag tcgctaaaac actttattatttagatttcc atcatctacc tcaaagtaaa 1920 aaagacctcg aagaatataa agaaaaacacaaaaacaagt ttattaacga aataaaacct 1980 gctacaccag caagtcaagc aaaaccagatcaagcaaaaa atgaaaaaga agtaaaacct 2040 gaatcagccc aagcagaatc ttcatcttcaaattctaatg attctaatag taaaaccact 2100 tcttcttcaa gtatgatggc gggtacaacccaaacaaata attcctctac agaaacaaca 2160 aattcaaatt cagcaacaac aacttcaacaacaacacaag cagcagcaac ttcagcctct 2220 tcggctaaag taaaaacaac taaattccaagaacaagtaa aagaacaaga acaaaaacaa 2280 gaaaaagcaa aagaaactaa ccaattattagatactaaaa gaaataaaga agactcaggg 2340 cttggattaa ttctttggga tttcctagtaaattcaaaat ataaaactct accaggaact 2400 acctgagatt tccatgttga accagataatttcaatgatc gtctaaaaat aacagcgatt 2460 ctaaaagaaa atacatccca ggcaaagtcaaatccagata gtaaaaacct aacttcccta 2520 tcgcgaaacc ttataataaa aggggttatggctaataaat acattgacta cttagtccaa 2580 gaagatccag tacttcttgt agattatacaagaagaaacc agattaaaac cgaaagagaa 2640 ggacaactaa tttgaaatca gttagcttcccctcaaatgg catctcctga aactagtccc 2700 gaaaaggcta agctcgagat caccgaggaaggactccgtg ttaaaaaagg tggcactaag 2760 ataaaagaga caagaaaaag cacaaccagcaatgctaaaa gcaatactaa ctccaaacca 2820 aataaaaagt tagtcctact aaaagggtctataaaaaacc cgggaacaaa aaaggaatga 2880 attcttgtag gatctgggaa taacgccaccaaaaacggaa gctccagcaa caactccaat 2940 acccaaatat gaataaccag actaggaacatctgttggtt cattaaaaac cgaaggtgag 3000 acagtccttg gaatttcaaa taataattcccaaggtgaag ttctctgaac tactattaaa 3060 tccaaactcg aaaacgaaaa tcaatcagataacaatcaaa tccaatactc cccaagtacg 3120 catagtttaa caaccaattc tcgatcaaatacccaacaat cagggcgaaa tcaaattaaa 3180 attacaaaca ctcaaagaaa aacaactacttcgccggccc aaagcccaat acaaaatcct 3240 gatccgaacc aaattgatgt aagacttggtctactagtac aagacaaaaa acttcatctt 3300 tggtggattg ctaatgatag ctctgatgagcctgagcata taacaattga tttcgctgaa 3360 gggacaaaat ttaattatga tgatttaaattatgtcggag ggcttttaaa aaatactaca 3420 aataatacca atacccaagc ccaagacgatgaaggtgatg gatatctggc cctaaaagga 3480 ttagggatct atgaatttcc tgatgatgaaagtattgatc aagccgctac tgttgaaaaa 3540 gcagagagat tatataaaca ctttatggggctatttaggg aa 3582 6 1194 PRT Mycoplasma hyopneumoniae 6 Met Asn Gln PheAsp Glu Lys Glu Lys Gln His Asn Lys Ala Lys Ala 1 5 10 15 Ile Leu SerThr Gly Phe Ser Val Thr Ser Ile Ala Thr Thr Val Val 20 25 30 Ala Val ProIle Gly Leu Thr Ile Phe Glu Lys Ser Phe Ser Ser Gln 35 40 45 Val Ser GlyGly Val Asp Lys Asn Lys Val Val Asp Leu Lys Ser Asp 50 55 60 Ser Asp GlnIle Phe Ser Glu Glu Asp Phe Ile Arg Ala Val Glu Asn 65 70 75 80 Leu LysLeu Phe Asp Lys Tyr Arg His Leu Thr Ala Arg Met Ala Leu 85 90 95 Gly LeuAla Arg Glu Ala Ala Asn Ala Phe Asn Phe Leu Asp Thr Tyr 100 105 110 AspTyr Thr Pro Ile Thr Lys His Ser Phe Lys Ile Ser Leu Asp Ile 115 120 125Ser Asp Ala Phe Ala Ala Asn Lys Glu Val Lys Ala Val Val Val Ser 130 135140 Ala Tyr Ser Gln Lys Tyr Gln Val Thr Tyr Ser Arg Leu Thr Ser Leu 145150 155 160 Lys Gly Trp Lys Glu Glu Asp Asp Phe Gly Asp Asp Ile Ile AspTyr 165 170 175 Gln Ile Asn Gln Glu Leu Ser Gly Leu Ser Leu Ser Ser LeuAla Pro 180 185 190 Glu Ser Ala His Leu Leu Ala Ser Glu Met Ala Phe ArgLeu Asp Asn 195 200 205 Asp Phe Gln Val Ala Tyr Lys Lys Thr Gly Ser ArgAla Glu Ala Phe 210 215 220 Arg Gln Ala Leu Ile Lys Asn Tyr Leu Gly TyrAsn Leu Val Asn Arg 225 230 235 240 Gln Gly Leu Pro Thr Met Leu Gln LysGly Tyr Val Leu Ala Pro Lys 245 250 255 Thr Ile Glu Asn Lys Asn Ala SerGlu Glu Lys Leu Val Asn Ile Asn 260 265 270 Glu Asn Asp Arg Ala Arg ValAsn Lys Leu Gln Lys Val Glu Asn Leu 275 280 285 Ala Phe Lys Asn Leu SerAsp Pro Asn Gly Thr Leu Ser Ile Thr Phe 290 295 300 Glu Leu Trp Asp ProAsn Gly Lys Leu Val Ser Glu Tyr Asp Phe Lys 305 310 315 320 Ile Lys GlyIle Lys Lys Leu Asp Phe Asp Leu Lys Lys Gln Glu Glu 325 330 335 Lys ValLeu Gln Lys Val Thr Glu Phe Val Glu Ile Lys Pro Tyr Val 340 345 350 GlnLeu Gly Leu Ile Arg Asp Asn Leu Ser Leu Ser Glu Ile Ile Tyr 355 360 365Lys Ser Asp Asn Asn Pro Glu Tyr Leu Arg Lys Ile Leu Ala Lys Leu 370 375380 Lys Glu His Asn Asn Asn Lys Arg Val Asp Asn Asn Thr Ser Thr Thr 385390 395 400 Lys Phe Gln Glu Glu Asp Leu Lys Asn Glu Pro Asn Ser Asn GlySer 405 410 415 Glu Gln Asp Ser Phe Glu Lys Ala Lys Glu Asn Phe Leu SerPhe Phe 420 425 430 Asp Leu Arg Ser Arg Leu Ile Pro Ile Pro Asp Leu ProLeu Tyr Tyr 435 440 445 Leu Lys Val Asn Ser Ile Asn Phe Asp Arg Asn IleGlu Glu Asn Glu 450 455 460 Lys Glu Lys Leu Leu Lys Asn Glu Gln Val ValLeu Lys Val Asp Phe 465 470 475 480 Ser Leu Lys Lys Val Val Ser Asp IleArg Ala Pro Tyr Leu Val Ser 485 490 495 Ser Gln Val Arg Ser Asn Tyr ProPro Val Leu Lys Ala Ser Leu Ala 500 505 510 Lys Ile Gly Lys Gly Ser AsnSer Lys Val Val Leu Leu Asp Leu Gly 515 520 525 Asn Leu Ser Ser Arg PheLys Val Gln Leu Asp Tyr Ser Ala Lys Gln 530 535 540 Arg Glu Ile Ile AsnThr Leu Leu Lys Glu Asn Pro Glu Arg Glu Lys 545 550 555 560 Glu Leu GlnAla Lys Ile Glu Ser Lys Thr Phe Ser Pro Ile Asp Leu 565 570 575 Asn AsnAsp Asp Leu Leu Ala Ile Glu Phe Gln Tyr Glu Asp Asn Pro 580 585 590 GluGly Asp Trp Ile Thr Leu Gly Arg Met Glu Lys Leu Val Lys Glu 595 600 605Val Ile Gln Tyr Lys Lys Glu Gly Lys Thr Phe Leu Asp Asp Glu Val 610 615620 Ala Lys Thr Leu Tyr Tyr Leu Asp Phe His His Leu Pro Gln Ser Lys 625630 635 640 Lys Asp Leu Glu Glu Tyr Lys Glu Lys His Lys Asn Lys Phe IleAsn 645 650 655 Glu Ile Lys Pro Ala Thr Pro Ala Ser Gln Ala Lys Pro AspGln Ala 660 665 670 Lys Asn Glu Lys Glu Val Lys Pro Glu Ser Ala Gln AlaGlu Ser Ser 675 680 685 Ser Ser Asn Ser Asn Asp Ser Asn Ser Lys Thr ThrSer Ser Ser Ser 690 695 700 Met Met Ala Gly Thr Thr Gln Thr Asn Asn SerSer Thr Glu Thr Thr 705 710 715 720 Asn Ser Asn Ser Ala Thr Thr Thr SerThr Thr Thr Gln Ala Ala Ala 725 730 735 Thr Ser Ala Ser Ser Ala Lys ValLys Thr Thr Lys Phe Gln Glu Gln 740 745 750 Val Lys Glu Gln Glu Gln LysGln Glu Lys Ala Lys Glu Thr Asn Gln 755 760 765 Leu Leu Asp Thr Lys ArgAsn Lys Glu Asp Ser Gly Leu Gly Leu Ile 770 775 780 Leu Trp Asp Phe LeuVal Asn Ser Lys Tyr Lys Thr Leu Pro Gly Thr 785 790 795 800 Thr Trp AspPhe His Val Glu Pro Asp Asn Phe Asn Asp Arg Leu Lys 805 810 815 Ile ThrAla Ile Leu Lys Glu Asn Thr Ser Gln Ala Lys Ser Asn Pro 820 825 830 AspSer Lys Asn Leu Thr Ser Leu Ser Arg Asn Leu Ile Ile Lys Gly 835 840 845Val Met Ala Asn Lys Tyr Ile Asp Tyr Leu Val Gln Glu Asp Pro Val 850 855860 Leu Leu Val Asp Tyr Thr Arg Arg Asn Gln Ile Lys Thr Glu Arg Glu 865870 875 880 Gly Gln Leu Ile Trp Asn Gln Leu Ala Ser Pro Gln Met Ala SerPro 885 890 895 Glu Thr Ser Pro Glu Lys Ala Lys Leu Glu Ile Thr Glu GluGly Leu 900 905 910 Arg Val Lys Lys Gly Gly Thr Lys Ile Lys Glu Thr ArgLys Ser Thr 915 920 925 Thr Ser Asn Ala Lys Ser Asn Thr Asn Ser Lys ProAsn Lys Lys Leu 930 935 940 Val Leu Leu Lys Gly Ser Ile Lys Asn Pro GlyThr Lys Lys Glu Trp 945 950 955 960 Ile Leu Val Gly Ser Gly Asn Asn AlaThr Lys Asn Gly Ser Ser Ser 965 970 975 Asn Asn Ser Asn Thr Gln Ile TrpIle Thr Arg Leu Gly Thr Ser Val 980 985 990 Gly Ser Leu Lys Thr Glu GlyGlu Thr Val Leu Gly Ile Ser Asn Asn 995 1000 1005 Asn Ser Gln Gly GluVal Leu Trp Thr Thr Ile Lys Ser Lys Leu Glu 1010 1015 1020 Asn Glu AsnGln Ser Asp Asn Asn Gln Ile Gln Tyr Ser Pro Ser Thr 1025 1030 1035 1040His Ser Leu Thr Thr Asn Ser Arg Ser Asn Thr Gln Gln Ser Gly Arg 10451050 1055 Asn Gln Ile Lys Ile Thr Asn Thr Gln Arg Lys Thr Thr Thr SerPro 1060 1065 1070 Ala Gln Ser Pro Ile Gln Asn Pro Asp Pro Asn Gln IleAsp Val Arg 1075 1080 1085 Leu Gly Leu Leu Val Gln Asp Lys Lys Leu HisLeu Trp Trp Ile Ala 1090 1095 1100 Asn Asp Ser Ser Asp Glu Pro Glu HisIle Thr Ile Asp Phe Ala Glu 1105 1110 1115 1120 Gly Thr Lys Phe Asn TyrAsp Asp Leu Asn Tyr Val Gly Gly Leu Leu 1125 1130 1135 Lys Asn Thr ThrAsn Asn Thr Asn Thr Gln Ala Gln Asp Asp Glu Gly 1140 1145 1150 Asp GlyTyr Leu Ala Leu Lys Gly Leu Gly Ile Tyr Glu Phe Pro Asp 1155 1160 1165Asp Glu Ser Ile Asp Gln Ala Ala Thr Val Glu Lys Ala Glu Arg Leu 11701175 1180 Tyr Lys His Phe Met Gly Leu Phe Arg Glu 1185 1190 7 5636 DNAMycoplasma hyopneumoniae 7 atgaaaaaca aaaaatcaac attactatta gccacagcggcagcaattat tggttcaact 60 gtttttggaa cagttgttgg tttggcttca aaagttaaatatcggggtgt aaatccaact 120 caaggagtaa tatctcaatt aggactgatt gattctgttgcatttaaacc ttcgattgca 180 aattttacaa gcgattatca aagtgttaaa aaagcacttttaaatgggaa aacctttgat 240 ccaaaaagtt cagaatttac tgattttgtc tcaaaatttgactttttgac taataatggg 300 agaaccgttt tggagatccc gaaaaaatat caggtggttatctcggaatt tagccccgag 360 gatgataaag aacgttttcg tcttggattt catctaaaagaaaaacttga agatggaaat 420 atagctcaat cagcaactaa atttatttat cttttaccacttgatatgcc caaagcggcc 480 ctgggtcaat attcttatat cgttgataaa aattttaataatttaattat ccatccttta 540 tctaattttt ctgctcaatc aataaagccg cttgcactgacccgttcaag tgattttata 600 gcaaaactta atcagtttaa caatcaggac gagctttgagtttatctgga aaaattcttt 660 gatcttgaag ctctaaaagc aaatattcgc ttacagacagccgattttag ttttgaaaaa 720 ggcaatttag ttgatccttt tgtttattct tttattagaaatccgcaaaa tcaaaaagaa 780 tgagctagtg atcttaatca agatcaaaaa actgtcagactttatcttcg aaccgaattt 840 agtcctcagg ctaaaaccat tttaaaagac tataaatacaaagatgagac tttcttaagt 900 agtatcgatt taaaagcaag taatggaact agtttatttgctaatgaaaa tgatctaaaa 960 gatcaattag atgttgatct tttagatgtc tctgattattttggaggcca atcagagaca 1020 attactagta attcccaagt taaacctgtc cctgctagtgagagatcttt aaaagaccgg 1080 gttaaattta aaaaagatca gcaaaaacca agaattgagaaatttagttt atatgaatat 1140 gatgctctaa gtttttattc ccaacttcaa gaattagtttctaaacctaa ttcaattaaa 1200 gatttagtta atgcaacttt agctcgtaat cttcggttttcattaggaaa atataatttt 1260 ctttttgatg atttagccag tcatcttgat tatacttttttagtttcaaa agcaaaaatt 1320 aaacaaagtt caattacaaa aaaattattc attgaattaccaatcaaaat tagtcttaaa 1380 tcttcaattt taggtgatca agaacctaat attaaaactttattcgaaaa agaagtgact 1440 tttaaattag ataacttccg tgatgttgaa atcgaaaaagcttttggact tttatatcca 1500 ggtgttaatg aagaacttga acaagcccga aaagctcaaagagcaagctt tgaaaaagaa 1560 aaatcgaaaa aaggtcttaa agaatttagt caacaaaaagaagaaaattc aaagcgataa 1620 acaatcaaga gggtcttgaa gaagatgata atattactgaaagacttcct gagaattccc 1680 cgattcaata tcagcaagaa aatgccggtt taggtgcaagtccggataaa ccttatatga 1740 taaaggatgt ccaaaatcaa cgttattatc tagcaaaatcacaaattcaa gaactaatta 1800 aggccaaaga ttataccaaa ttagccaaac ttttatccaatagacatact tataatattt 1860 ctttaagatt aaaagaacaa ctttttgatg taaatccaagaattccgagc tctagagata 1920 tagaaaaggc aaaatttgtt cttgataaaa ccgaaaagaataaatactgg cagatttatt 1980 caagtgcttc tcctgttttc caaaataaat gatcactttttggatattac cgttatttat 2040 taggtcttga tccaaaacaa acaatccacg aattagtaaaattaggacaa aaagcgggtc 2100 ttcaatttga aggatatgaa aatcttcctt ctgatttcaatcttgaggat cttaagaata 2160 ttaggattaa aacaccttta tttagtcaaa aagataatttcaaattatct ttacttgatt 2220 ttaataatta ttatgacggt gaaattaaag ccccagaatttggtcttcct ttatttttgc 2280 caaaagaatt aagaagaaat agttcaaatt ctggtggttctcaaaactct aatagccctt 2340 gagaacaaga aattattagc caatttaaag atcaaaatctatctaatcag gatcagttag 2400 cccagtttag tactaaaatc tgggaaaaaa tcattggtgatgaaaacgaa tttgatcaaa 2460 ataacagact tcagtataaa cttttaaaag atcttcaagaatcttggatt aataaaaccc 2520 gcgataatct ttattggact tatctaggtg ataaacttaaagttaaacca aaaaataatt 2580 tagaggctaa atttagacaa atttccaatt tacaagagcttttaactgct ttttatactt 2640 cagctgctct ttctaataac tgaaattatt atcaagattcaggagcaaag tcaactatta 2700 tttttgaaga aatagctgag ctagatccaa aagtaaaagaaaaagttgga gctgatgttt 2760 atcaattaaa attccattat gcaatcggtt ttgatgataatgctggtaag tttaatcaag 2820 aagtaattcg ttcttcaagt agaacaattt atcttaaaacctcagggaaa tccaaattag 2880 aagcagatac aattgatcaa cttaatcaag cagttaaaaatgcaccttta ggtcttcaaa 2940 gtttttatct tgatactgaa agatttgggg ttttccaaaaattagccact tccttagcag 3000 ttcaacataa acaaaaagaa aaaacactac ctaaaaaactaaataatgat ggctatactt 3060 taattcatga taaacttaaa aaaccagtaa ttccccaaattagttcaagt ccagaaaaag 3120 actgatttga aggtaaatta aaccaaaacg ggcaaagccaaaatgtaaat gtctcaactt 3180 ttggctcaat aatcgagtcc ccttatttta gtactaatttccaagaagat gctgacttag 3240 accaggatgg acaagatgat tcaagacaag gaaataatagtctagataat caagaagcag 3300 gtcttttaaa acaaaaactg gcaattttat taggtaatcaatttatccaa tattatcaac 3360 aaaatgataa agaaattgaa ttcgagatta tcaatgttgagaaagtttca gagcttagtt 3420 tccgcgttga atttaaatta gcaaaaactc ttgaagacaacggaaaaact attcgagttt 3480 tatcagatga gacaatgtca ttaattgtta atactacaattgaaaaaaca ccagaaatga 3540 gtgcggttcc cgaagtattt gatactaaat gggttgagcaatatgatcca agaaccccgc 3600 ttgcggcaaa gacaaagttt gtcttaaaat tcaaagatcaaataccagtg gatggcagtg 3660 gaaatatttc tgataaatga ctagcaagta ttcctttggtgattcaccaa caaatgttgc 3720 gtcttagtcc tgtggttaaa acgataagag agctcggtctaaagaccgaa caacaacaac 3780 aacaacaaca acaacaacaa caacaacaac cccaaaagaaagctgttaga aaagaggaag 3840 aactagaaac ctataatcca aaagacgagt ttaatattcttaatcctttg acaaaagctc 3900 accgccttac cttatcaaat ttggtaaata atgatccaaattataaaatt gaagatttaa 3960 aagtaatcaa aaatgaagct ggtgaccatc aattagcattttctctaaga gctaataata 4020 tcaaaagatt aatgaataca ccaattactt ttgctgattataatcccttt ttctattata 4080 atgaagactg aagaagtata gataaatatt taaataataaaggaaatgtg agttctcacc 4140 aacaacaagc agccgggggt aatcaaggct cgggtctaatccaaagactt aataaaaata 4200 ttaagcccga aacttttacc cccgcactca tagctcttaaacgagataat aatactaatc 4260 tttctaacta ttctgataaa ataataatga tcaaaccaaaatatttggtt gaacgatcaa 4320 ttggtgttcc ctgatcaacc ggccttgatg gttatattggttcagaacaa accaaggacg 4380 gaacttcctc aagcagtcaa caaaagggat ttaagcaagattttattcag gctttaggtc 4440 ttaaaaacac tgaatatcat ggtaaactag gtctttcaattagaattttt gatcctggaa 4500 atgaactagc aaaaattaag gatgcttcaa ataaaaaaggggaagaaaag ctgttaaaat 4560 catatgattt atttaaaaac tatttaaatg aatatgagaaaaaatcccct aaaattgcta 4620 agggatgaac aaatattcat cctgatcaaa aagaatatccaaatccaaat caaaaactac 4680 ctgaaaatta tcttaaccta gttttaaatc aaccttgaaaggttacttta tataattcaa 4740 gtgattttat tactaattta tttgttgaac ctgaaggctcagatcgtgga tcaggaacaa 4800 aattaaaaca agtaatccag aagcaagtta ataataactatgctgactgg gggtctgcat 4860 atctcacgtt ctggtatgat aaaaatatca ttaccaatcagccaaatgtt ataactgcaa 4920 acattgctga tgtctttatt aaagatgtaa aagaacttgaagataataca aaactaattg 4980 ctccaaatat tactcaatga tggccaaata ttagcggctcaaaagagaaa ttttataagc 5040 caacagtgtt ttttggtaat tgagaaaatg aaaacagcagtatgaattcc caggcgcaga 5100 cccctacctg ggagaagatc agagaaggat ttgctctccaagcgcttaaa tccagctttg 5160 atcaaaaaac aaggacattt gtccttacaa caaatgctcctttaccttta tgaaaatacg 5220 gaccattagg tttccaaaat gggccgaatt tcaaaacacaagattgaagg cttgttttcc 5280 aaaatgatga taaccaaata gccgcgctaa gagtccaggagcaagatcgc ccagaaaaat 5340 caagcgaaga taaagacaag caaaaatgga ttaaatttaaagttgttatc cctgaagaaa 5400 tgtttaattc cggtaatata cgttttgttg gggtaatgcagatccaaggt cctaatactt 5460 tatgacttcc agtgattaat tcttcggtta tctatgacttctatcgcgga acaggagatt 5520 ctaatgatgt cgccaatctt aatgtagctc cttgacaggttaaaacaatc gcatttacaa 5580 ataacgcctt taataatgtt ttcaaagagt ttaatatctctaaaaaaata gtagaa 5636 8 1879 PRT Mycoplasma hyopneumoniae 8 Met Lys AsnLys Lys Ser Thr Leu Leu Leu Ala Thr Ala Ala Ala Ile 1 5 10 15 Ile GlySer Thr Val Phe Gly Thr Val Val Gly Leu Ala Ser Lys Val 20 25 30 Lys TyrArg Gly Val Asn Pro Thr Gln Gly Val Ile Ser Gln Leu Gly 35 40 45 Leu IleAsp Ser Val Ala Phe Lys Pro Ser Ile Ala Asn Phe Thr Ser 50 55 60 Asp TyrGln Ser Val Lys Lys Ala Leu Leu Asn Gly Lys Thr Phe Asp 65 70 75 80 ProLys Ser Ser Glu Phe Thr Asp Phe Val Ser Lys Phe Asp Phe Leu 85 90 95 ThrAsn Asn Gly Arg Thr Val Leu Glu Ile Pro Lys Lys Tyr Gln Val 100 105 110Val Ile Ser Glu Phe Ser Pro Glu Asp Asp Lys Glu Arg Phe Arg Leu 115 120125 Gly Phe His Leu Lys Glu Lys Leu Glu Asp Gly Asn Ile Ala Gln Ser 130135 140 Ala Thr Lys Phe Ile Tyr Leu Leu Pro Leu Asp Met Pro Lys Ala Ala145 150 155 160 Leu Gly Gln Tyr Ser Tyr Ile Val Asp Lys Asn Phe Asn AsnLeu Ile 165 170 175 Ile His Pro Leu Ser Asn Phe Ser Ala Gln Ser Ile LysPro Leu Ala 180 185 190 Leu Thr Arg Ser Ser Asp Phe Ile Ala Lys Leu AsnGln Phe Asn Asn 195 200 205 Gln Asp Glu Leu Trp Val Tyr Leu Glu Lys PhePhe Asp Leu Glu Ala 210 215 220 Leu Lys Ala Asn Ile Arg Leu Gln Thr AlaAsp Phe Ser Phe Glu Lys 225 230 235 240 Gly Asn Leu Val Asp Pro Phe ValTyr Ser Phe Ile Arg Asn Pro Gln 245 250 255 Asn Gln Lys Glu Trp Ala SerAsp Leu Asn Gln Asp Gln Lys Thr Val 260 265 270 Arg Leu Tyr Leu Arg ThrGlu Phe Ser Pro Gln Ala Lys Thr Ile Leu 275 280 285 Lys Asp Tyr Lys TyrLys Asp Glu Thr Phe Leu Ser Ser Ile Asp Leu 290 295 300 Lys Ala Ser AsnGly Thr Ser Leu Phe Ala Asn Glu Asn Asp Leu Lys 305 310 315 320 Asp GlnLeu Asp Val Asp Leu Leu Asp Val Ser Asp Tyr Phe Gly Gly 325 330 335 GlnSer Glu Thr Ile Thr Ser Asn Ser Gln Val Lys Pro Val Pro Ala 340 345 350Ser Glu Arg Ser Leu Lys Asp Arg Val Lys Phe Lys Lys Asp Gln Gln 355 360365 Lys Pro Arg Ile Glu Lys Phe Ser Leu Tyr Glu Tyr Asp Ala Leu Ser 370375 380 Phe Tyr Ser Gln Leu Gln Glu Leu Val Ser Lys Pro Asn Ser Ile Lys385 390 395 400 Asp Leu Val Asn Ala Thr Leu Ala Arg Asn Leu Arg Phe SerLeu Gly 405 410 415 Lys Tyr Asn Phe Leu Phe Asp Asp Leu Ala Ser His LeuAsp Tyr Tyr 420 425 430 Phe Leu Val Ser Lys Ala Lys Ile Lys Gln Ser SerIle Thr Lys Lys 435 440 445 Leu Phe Ile Glu Leu Pro Ile Lys Ile Ser LeuLys Ser Ser Ile Leu 450 455 460 Gly Asp Gln Glu Pro Asn Ile Lys Thr LeuPhe Glu Lys Glu Val Thr 465 470 475 480 Phe Lys Leu Asp Asn Phe Arg AspVal Glu Ile Glu Lys Ala Phe Gly 485 490 495 Leu Leu Tyr Pro Gly Val AsnGlu Glu Leu Glu Gln Ala Arg Lys Ala 500 505 510 Gln Arg Ala Ser Phe GluLys Glu Lys Ser Lys Lys Gly Leu Lys Glu 515 520 525 Phe Ser Gln Gln LysGlu Glu Asn Ser Lys Ala Ile Asn Asn Gln Glu 530 535 540 Gly Leu Glu GluAsp Asp Asn Ile Thr Glu Arg Leu Pro Glu Asn Ser 545 550 555 560 Pro IleGln Tyr Gln Gln Glu Asn Ala Gly Leu Gly Ala Ser Pro Asp 565 570 575 LysPro Tyr Met Ile Lys Asp Val Gln Asn Gln Arg Tyr Tyr Leu Ala 580 585 590Lys Ser Gln Ile Gln Glu Leu Ile Lys Ala Lys Asp Tyr Thr Lys Leu 595 600605 Ala Lys Leu Leu Ser Asn Arg His Thr Tyr Asn Ile Ser Leu Arg Leu 610615 620 Lys Glu Gln Leu Phe Asp Val Asn Pro Arg Ile Pro Ser Ser Arg Asp625 630 635 640 Ile Glu Lys Ala Lys Phe Val Leu Asp Lys Thr Glu Lys AsnLys Tyr 645 650 655 Trp Gln Ile Tyr Ser Ser Ala Ser Pro Val Phe Gln AsnLys Trp Ser 660 665 670 Leu Phe Gly Tyr Tyr Arg Tyr Leu Leu Gly Leu AspPro Lys Gln Thr 675 680 685 Ile His Glu Leu Val Lys Leu Gly Gln Lys AlaGly Leu Gln Phe Glu 690 695 700 Gly Tyr Glu Asn Leu Pro Ser Asp Phe AsnLeu Glu Asp Leu Lys Asn 705 710 715 720 Ile Arg Ile Lys Thr Pro Leu PheSer Gln Lys Asp Asn Phe Lys Leu 725 730 735 Ser Leu Leu Asp Phe Asn AsnTyr Tyr Asp Gly Glu Ile Lys Ala Pro 740 745 750 Glu Phe Gly Leu Pro LeuPhe Leu Pro Lys Glu Leu Arg Arg Asn Ser 755 760 765 Ser Asn Ser Gly GlySer Gln Asn Ser Asn Ser Pro Trp Glu Gln Glu 770 775 780 Ile Ile Ser GlnPhe Lys Asp Gln Asn Leu Ser Asn Gln Asp Gln Leu 785 790 795 800 Ala GlnPhe Ser Thr Lys Ile Trp Glu Lys Ile Ile Gly Asp Glu Asn 805 810 815 GluPhe Asp Gln Asn Asn Arg Leu Gln Tyr Lys Leu Leu Lys Asp Leu 820 825 830Gln Glu Ser Trp Ile Asn Lys Thr Arg Asp Asn Leu Tyr Trp Thr Tyr 835 840845 Leu Gly Asp Lys Leu Lys Val Lys Pro Lys Asn Asn Leu Glu Ala Lys 850855 860 Phe Arg Gln Ile Ser Asn Leu Gln Glu Leu Leu Thr Ala Phe Tyr Thr865 870 875 880 Ser Ala Ala Leu Ser Asn Asn Trp Asn Tyr Tyr Gln Asp SerGly Ala 885 890 895 Lys Ser Thr Ile Ile Phe Glu Glu Ile Ala Glu Leu AspPro Lys Val 900 905 910 Lys Glu Lys Val Gly Ala Asp Val Tyr Gln Leu LysPhe His Tyr Ala 915 920 925 Ile Gly Phe Asp Asp Asn Ala Gly Lys Phe AsnGln Glu Val Ile Arg 930 935 940 Ser Ser Ser Arg Thr Ile Tyr Leu Lys ThrSer Gly Lys Ser Lys Leu 945 950 955 960 Glu Ala Asp Thr Ile Asp Gln LeuAsn Gln Ala Val Lys Asn Ala Pro 965 970 975 Leu Gly Leu Gln Ser Phe TyrLeu Asp Thr Glu Arg Phe Gly Val Phe 980 985 990 Gln Lys Leu Ala Thr SerLeu Ala Val Gln His Lys Gln Lys Glu Lys 995 1000 1005 Thr Leu Pro LysLys Leu Asn Asn Asp Gly Tyr Thr Leu Ile His Asp 1010 1015 1020 Lys LeuLys Lys Pro Val Ile Pro Gln Ile Ser Ser Ser Pro Glu Lys 1025 1030 10351040 Asp Trp Phe Glu Gly Lys Leu Asn Gln Asn Gly Gln Ser Gln Asn Val1045 1050 1055 Asn Val Ser Thr Phe Gly Ser Ile Ile Glu Ser Pro Tyr PheSer Thr 1060 1065 1070 Asn Phe Gln Glu Asp Ala Asp Leu Asp Gln Asp GlyGln Asp Asp Ser 1075 1080 1085 Arg Gln Gly Asn Asn Ser Leu Asp Asn GlnGlu Ala Gly Leu Leu Lys 1090 1095 1100 Gln Lys Leu Ala Ile Leu Leu GlyAsn Gln Phe Ile Gln Tyr Tyr Gln 1105 1110 1115 1120 Gln Asn Asp Lys GluIle Glu Phe Glu Ile Ile Asn Val Glu Lys Val 1125 1130 1135 Ser Glu LeuSer Phe Arg Val Glu Phe Lys Leu Ala Lys Thr Leu Glu 1140 1145 1150 AspAsn Gly Lys Thr Ile Arg Val Leu Ser Asp Glu Thr Met Ser Leu 1155 11601165 Ile Val Asn Thr Thr Ile Glu Lys Thr Pro Glu Met Ser Ala Val Pro1170 1175 1180 Glu Val Phe Asp Thr Lys Trp Val Glu Gln Tyr Asp Pro ArgThr Pro 1185 1190 1195 1200 Leu Ala Ala Lys Thr Lys Phe Val Leu Lys PheLys Asp Gln Ile Pro 1205 1210 1215 Val Asp Gly Ser Gly Asn Ile Ser AspLys Trp Leu Ala Ser Ile Pro 1220 1225 1230 Leu Val Ile His Gln Gln MetLeu Arg Leu Ser Pro Val Val Lys Thr 1235 1240 1245 Ile Arg Glu Leu GlyLeu Lys Thr Glu Gln Gln Gln Gln Gln Gln Gln 1250 1255 1260 Gln Gln GlnGln Gln Gln Pro Gln Lys Lys Ala Val Arg Lys Glu Glu 1265 1270 1275 1280Glu Leu Glu Thr Tyr Asn Pro Lys Asp Glu Phe Asn Ile Leu Asn Pro 12851290 1295 Leu Thr Lys Ala His Arg Leu Thr Leu Ser Asn Leu Val Asn AsnAsp 1300 1305 1310 Pro Asn Tyr Lys Ile Glu Asp Leu Lys Val Ile Lys AsnGlu Ala Gly 1315 1320 1325 Asp His Gln Leu Ala Phe Ser Leu Arg Ala AsnAsn Ile Lys Arg Leu 1330 1335 1340 Met Asn Thr Pro Ile Thr Phe Ala AspTyr Asn Pro Phe Phe Tyr Tyr 1345 1350 1355 1360 Asn Glu Asp Trp Arg SerIle Asp Lys Tyr Leu Asn Asn Lys Gly Asn 1365 1370 1375 Val Ser Ser HisGln Gln Gln Ala Ala Gly Gly Asn Gln Gly Ser Gly 1380 1385 1390 Leu IleGln Arg Leu Asn Lys Asn Ile Lys Pro Glu Thr Phe Thr Pro 1395 1400 1405Ala Leu Ile Ala Leu Lys Asp Arg Asn Asn Thr Asn Leu Ser Asn Tyr 14101415 1420 Ser Asp Lys Ile Ile Met Ile Lys Pro Lys Tyr Leu Val Glu ArgSer 1425 1430 1435 1440 Ile Gly Val Pro Trp Ser Thr Gly Leu Asp Gly TyrIle Gly Ser Glu 1445 1450 1455 Gln Thr Lys Asp Gly Thr Ser Ser Ser SerGln Gln Lys Gly Phe Asp 1460 1465 1470 Gln Asp Phe Ile Gln Ala Leu GlyLeu Lys Asn Thr Glu Tyr His Gly 1475 1480 1485 Lys Leu Gly Leu Ser IleArg Ile Phe Asp Pro Gly Asn Glu Leu Ala 1490 1495 1500 Lys Ile Lys AspAla Ser Asn Lys Lys Gly Glu Glu Lys Leu Leu Lys 1505 1510 1515 1520 SerTyr Asp Leu Phe Lys Asn Tyr Leu Asn Glu Tyr Glu Lys Lys Ser 1525 15301535 Pro Lys Ile Ala Lys Gly Trp Thr Asn Ile His Pro Asp Gln Lys Glu1540 1545 1550 Tyr Pro Asn Pro Asn Gln Lys Leu Pro Glu Asn Tyr Leu AsnLeu Val 1555 1560 1565 Leu Asn Gln Pro Trp Lys Val Thr Leu Tyr Asn SerSer Asp Phe Ile 1570 1575 1580 Thr Asn Leu Phe Val Glu Pro Glu Gly SerAsp Arg Gly Ser Gly Thr 1585 1590 1595 1600 Lys Leu Lys Gln Val Ile GlnLys Gln Val Asn Asn Asn Tyr Ala Asp 1605 1610 1615 Trp Gly Ser Ala TyrLeu Thr Phe Trp Tyr Asp Lys Asn Ile Ile Thr 1620 1625 1630 Asn Gln ProAsn Val Ile Thr Ala Asn Ile Ala Asp Val Phe Ile Lys 1635 1640 1645 AspVal Lys Glu Leu Glu Asp Asn Thr Lys Leu Ile Ala Pro Asn Ile 1650 16551660 Thr Gln Trp Trp Pro Asn Ile Ser Gly Ser Lys Glu Lys Phe Tyr Lys1665 1670 1675 1680 Pro Thr Val Phe Phe Gly Asn Trp Glu Asn Glu Asn SerSer Met Asn 1685 1690 1695 Ser Gln Ala Gln Thr Pro Thr Trp Glu Lys IleArg Glu Gly Phe Ala 1700 1705 1710 Leu Gln Ala Leu Lys Ser Ser Phe AspGln Lys Thr Arg Thr Phe Val 1715 1720 1725 Leu Thr Thr Asn Ala Pro LeuPro Leu Trp Lys Tyr Gly Pro Leu Gly 1730 1735 1740 Phe Gln Asn Gly ProAsn Phe Lys Thr Gln Asp Trp Arg Leu Val Phe 1745 1750 1755 1760 Gln AsnAsp Asp Asn Gln Ile Ala Ala Leu Arg Val Gln Glu Gln Asp 1765 1770 1775Arg Pro Glu Lys Ser Ser Glu Asp Lys Asp Lys Gln Lys Trp Ile Lys 17801785 1790 Phe Lys Val Val Ile Pro Glu Glu Met Phe Asn Ser Gly Asn IleArg 1795 1800 1805 Phe Val Gly Val Met Gln Ile Gln Gly Pro Asn Thr LeuTrp Leu Pro 1810 1815 1820 Val Ile Asn Ser Ser Val Ile Tyr Asp Phe TyrArg Gly Thr Gly Asp 1825 1830 1835 1840 Ser Asn Asp Val Ala Asn Leu AsnVal Ala Pro Trp Gln Val Lys Thr 1845 1850 1855 Ile Ala Phe Thr Asn AsnAla Phe Asn Asn Val Phe Lys Glu Phe Asn 1860 1865 1870 Ile Ser Lys LysIle Val Glu 1875 9 3003 DNA Mycoplasma hyopneumoniae 9 ttgattttaattgaagaaat taaggaaatc aaaaaattta tggaaaacac caacttgcac 60 tacaaaaaaaaaaaaaaaaa aagcactaac ctttctagaa aaaatctttt aacaattggg 120 gccgcagtttttttcggaat tgcaataatc acaattccgc ttgtcaccgt tgctaattga 180 aagatcaaagatccacgact tcaagtacaa aatcaagcaa aattaattac aaatattcaa 240 ctaaaagatgagtatcaaaa tggaaattta agctattttg atcttaaaaa acagcttttt 300 aatgctgataatactaaaaa aactgggatt gactatagcc agttttttga tttttaccaa 360 aaaaataacacgagcctacc aattaatttt gccactgatt atggctgaaa tcgttacaaa 420 cttgatgtttttgatctaaa accacttgat caagaacaat cttttgaaat ttattatcgt 480 ttagtatatcaactacctga tgataaaaag gcaatttctg atcttttaac ccaaaaagtt 540 atctgaaattatctccctga ttattcactt gctaatttcg ctaatttttc aagttcaaaa 600 ttggaaaaactaagagctta taccaacaag gaatttagtt tatcaaccaa aaaagaactt 660 acaaaattagtaaaattaga agactttgaa aagcaagtaa actgggcaat aaataataat 720 gaagcccgcaaaattattaa taaatatttt aatttagaag aaattattgc cgagattctt 780 aataataaagaattttctta tctagatgaa agtggaatat gaaatccgca atatcagatt 840 gaacttgtaagagatcaaat tttaggtcag gattttttag caaaaacagg tcaaaaagga 900 atttataaattaacatttta tgctgctttt tcgccgaatt ttgctaaaaa aattgcggct 960 gatctcaataaaagttcaaa gtttcatttt ggaattaaca ttgatcttaa taatcttttc 1020 cttgataaaacagtcgctga aaatattaaa ataactgaat tttctgaaga tgattattac 1080 ccacaaataaattttgaaaa aaatttagaa gccgaaatta atggttgaga ttttctaaat 1140 tattacaataaccaaatttt tgcaactcaa aacgagagag aagattttct caagaacctt 1200 atagcaaaaattgttagaac tccgcttctg aaaaaagttg aatttgaaaa taaattatcc 1260 ggtattgattatgcaaaatt tttaaaatat ttaaaattag atattaaatt agatgctaat 1320 tcaactaaattggcttttaa aaataaccaa attgttgcca aaattttcgg aaaaattatt 1380 cttagaaatgctgaaaatca aattgtcgct gaaaaaaact tttcccaaac tattgaacat 1440 ctaaaccgtctcgggcaaaa tgatgctgaa ttagtaaagc aaattaaaca gacaaaattt 1500 gaatttaaaccagaaactag aaaaaaaatt gcaaaccaaa agggtgcgcc aaaatcagaa 1560 attcttgcactcttaaatgc caataaattt gataaattaa aaaatatcct tgaaaatggt 1620 gattattatggctatgaatt taacgaagat cgcttaaaat tattagttca taattcacaa 1680 ttacctaatgttgaagaatt tgcaaaatta agtgtagttc ctgagaaaat gtctgaggga 1740 attattaatctttggaataa gtcatttaaa acaaatcaag aggttagtac atttttatct 1800 ttacttgcaaaaagggatat cagttttgtt gcaaaatatt gatatgatct tttaaataaa 1860 tttaaattaattgatccaaa aacacaatgg cctgaaaatc ttgaccaaaa tagtttattt 1920 aaacatttaagtcaaataaa aattcagcct cctgagaaaa aagcagtttc actgacctcc 1980 gatttttgacttttttcatt aaataatgac tacctaattt cccctgatta tcttaataat 2040 agtttttaccttcactcaaa tttaaaaaat actttggact taatcaaaac tgaaagcgca 2100 tttaacacgagagattttgt cgaacatata agagaacttg caaaatcaat taaaccaaaa 2160 gattttatccaagaaaaagg taaaaatcca attacaaatc ttagtgaatt tctagttgct 2220 ttttattcgcttatttattc aaaggatcaa ggacttcttg ctgaatcact cgggcaaaat 2280 ttagactataaaattcagtt tgaactcgaa cctataagcc taaatgtagc agttagtcag 2340 gaaaaaactaatccaaataa taatttaaga ttaaataata atttaagatt aaaatattga 2400 tataaaattggttcagttga tcaaaatggg aatttaattc aagtgattta ccaaacaaaa 2460 aaagaaactttggatcttgt agttaatgaa aataataaat tgcttagtga agatgtagaa 2520 aaattaaatgaaattgctac taattttcca agtgcagacc aaattatttt ccttaaaaaa 2580 gaagattatacccaacttgt tgatagtata aaacaagtaa ttaaaacgga aaatactcca 2640 gttaaaattgataatcagat caaaaatcta ccttttagtc aattttttga aaataattac 2700 ccagattatggtttttatat aataaaaaca agtaaaaatt tagaaagtag taaacctgaa 2760 gcagcaaaagttgctgcaaa accttcagca gccaagccag tagcagctaa accagaacaa 2820 caagaaattcatcaaagcga agaaattccc ggagttctta ctaatacaat atctcaactt 2880 ggcaatcagatacgacataa ttttgattta tatgtataca aaaaagatca gccacagatt 2940 cactcaagtaagccagttag ggtaattatt attgaaagtt cagaatcact atttgcttta 3000 aaa 3003 101001 PRT Mycoplasma hyopneumoniae 10 Met Ile Leu Ile Glu Glu Ile Lys GluIle Lys Lys Phe Met Glu Asn 1 5 10 15 Thr Asn Leu His Tyr Lys Lys LysLys Lys Lys Ser Thr Asn Leu Ser 20 25 30 Arg Lys Asn Leu Leu Thr Ile GlyAla Ala Val Phe Phe Gly Ile Ala 35 40 45 Ile Ile Thr Ile Pro Leu Val ThrVal Ala Asn Trp Lys Ile Lys Asp 50 55 60 Pro Arg Leu Gln Val Gln Asn GlnAla Lys Leu Ile Thr Asn Ile Gln 65 70 75 80 Leu Lys Asp Glu Tyr Gln AsnGly Asn Leu Ser Tyr Phe Asp Leu Lys 85 90 95 Lys Gln Leu Phe Asn Ala AspAsn Thr Lys Lys Thr Gly Ile Asp Tyr 100 105 110 Ser Gln Phe Phe Asp PheTyr Gln Lys Asn Asn Thr Ser Leu Pro Ile 115 120 125 Asn Phe Ala Thr AspTyr Gly Trp Asn Arg Tyr Lys Leu Asp Val Phe 130 135 140 Asp Leu Lys ProLeu Asp Gln Glu Gln Ser Phe Glu Ile Tyr Tyr Arg 145 150 155 160 Leu ValTyr Gln Leu Pro Asp Asp Lys Lys Ala Ile Ser Asp Leu Leu 165 170 175 ThrGln Lys Val Ile Trp Asn Tyr Leu Pro Asp Tyr Ser Leu Ala Asn 180 185 190Phe Ala Asn Phe Ser Ser Ser Lys Leu Glu Lys Leu Arg Ala Tyr Thr 195 200205 Asn Lys Glu Phe Ser Leu Ser Thr Lys Lys Glu Leu Thr Lys Leu Val 210215 220 Lys Leu Glu Asp Phe Glu Lys Gln Val Asn Trp Ala Ile Asn Asn Asn225 230 235 240 Glu Ala Arg Lys Ile Ile Asn Lys Tyr Phe Asn Leu Glu GluIle Ile 245 250 255 Ala Glu Ile Leu Asn Asn Lys Glu Phe Ser Tyr Leu AspGlu Ser Gly 260 265 270 Ile Trp Asn Pro Gln Tyr Gln Ile Glu Leu Val ArgAsp Gln Ile Leu 275 280 285 Gly Gln Asp Phe Leu Ala Lys Thr Gly Gln LysGly Ile Tyr Lys Leu 290 295 300 Thr Phe Tyr Ala Ala Phe Ser Pro Asn PheAla Lys Lys Ile Ala Ala 305 310 315 320 Asp Leu Asn Lys Ser Ser Lys PheHis Phe Gly Ile Asn Ile Asp Leu 325 330 335 Asn Asn Leu Phe Leu Asp LysThr Val Ala Glu Asn Ile Lys Ile Thr 340 345 350 Glu Phe Ser Glu Asp AspTyr Tyr Pro Gln Ile Asn Phe Glu Lys Asn 355 360 365 Leu Glu Ala Glu IleAsn Gly Trp Asp Phe Leu Asn Tyr Tyr Asn Asn 370 375 380 Gln Ile Phe AlaThr Gln Asn Glu Arg Glu Asp Phe Leu Lys Asn Leu 385 390 395 400 Ile AlaLys Ile Val Arg Thr Pro Leu Leu Lys Lys Val Glu Phe Glu 405 410 415 AsnLys Leu Ser Gly Ile Asp Tyr Ala Lys Phe Leu Lys Tyr Leu Lys 420 425 430Leu Asp Ile Lys Leu Asp Ala Asn Ser Thr Lys Leu Ala Phe Lys Asn 435 440445 Asn Gln Ile Val Ala Lys Ile Phe Gly Lys Ile Ile Leu Arg Asn Ala 450455 460 Glu Asn Gln Ile Val Ala Glu Lys Asn Phe Ser Gln Thr Ile Glu His465 470 475 480 Leu Asn Arg Leu Gly Gln Asn Asp Ala Glu Leu Val Lys GlnIle Lys 485 490 495 Gln Thr Lys Phe Glu Phe Lys Pro Glu Thr Arg Lys LysIle Ala Asn 500 505 510 Gln Lys Gly Ala Pro Lys Ser Glu Ile Leu Ala LeuLeu Asn Ala Asn 515 520 525 Lys Phe Asp Lys Leu Lys Asn Ile Leu Glu AsnGly Asp Tyr Tyr Gly 530 535 540 Tyr Glu Phe Asn Glu Asp Arg Leu Lys LeuLeu Val His Asn Ser Gln 545 550 555 560 Leu Pro Asn Val Glu Glu Phe AlaLys Leu Ser Val Val Pro Glu Lys 565 570 575 Met Ser Glu Gly Ile Ile AsnLeu Trp Asn Lys Ser Phe Lys Thr Asn 580 585 590 Gln Glu Val Ser Thr PheLeu Ser Leu Leu Ala Lys Arg Asp Ile Ser 595 600 605 Phe Val Ala Lys TyrTrp Tyr Asp Leu Leu Asn Lys Phe Lys Leu Ile 610 615 620 Asp Pro Lys ThrGln Trp Pro Glu Asn Leu Asp Gln Asn Ser Leu Phe 625 630 635 640 Lys HisLeu Ser Gln Ile Lys Ile Gln Pro Pro Glu Lys Lys Ala Val 645 650 655 SerLeu Thr Ser Asp Phe Trp Leu Phe Ser Leu Asn Asn Asp Tyr Leu 660 665 670Ile Ser Pro Asp Tyr Leu Asn Asn Ser Phe Tyr Leu His Ser Asn Leu 675 680685 Lys Asn Thr Leu Asp Leu Ile Lys Thr Glu Ser Ala Phe Asn Thr Arg 690695 700 Asp Phe Val Glu His Ile Arg Glu Leu Ala Lys Ser Ile Lys Pro Lys705 710 715 720 Asp Phe Ile Gln Glu Lys Gly Lys Asn Pro Ile Thr Asn LeuSer Glu 725 730 735 Phe Leu Val Ala Phe Tyr Ser Leu Ile Tyr Ser Lys AspGln Gly Leu 740 745 750 Leu Ala Glu Ser Leu Gly Gln Asn Leu Asp Tyr LysIle Gln Phe Glu 755 760 765 Leu Glu Pro Ile Ser Leu Asn Val Ala Val SerGln Glu Lys Thr Asn 770 775 780 Pro Asn Asn Asn Leu Arg Leu Asn Asn AsnLeu Arg Leu Lys Tyr Trp 785 790 795 800 Tyr Lys Ile Gly Ser Val Asp GlnAsn Gly Asn Leu Ile Gln Val Ile 805 810 815 Tyr Gln Thr Lys Lys Glu ThrLeu Asp Leu Val Val Asn Glu Asn Asn 820 825 830 Lys Leu Leu Ser Glu AspVal Glu Lys Leu Asn Glu Ile Ala Thr Asn 835 840 845 Phe Pro Ser Ala AspGln Ile Ile Phe Leu Lys Lys Glu Asp Tyr Thr 850 855 860 Gln Leu Val AspSer Ile Lys Gln Val Ile Lys Thr Glu Asn Thr Pro 865 870 875 880 Val LysIle Asp Asn Gln Ile Lys Asn Leu Pro Phe Ser Gln Phe Phe 885 890 895 GluAsn Asn Tyr Pro Asp Tyr Gly Phe Tyr Ile Ile Lys Thr Ser Lys 900 905 910Asn Leu Glu Ser Ser Lys Pro Glu Ala Ala Lys Val Ala Ala Lys Pro 915 920925 Ser Ala Ala Lys Pro Val Ala Ala Lys Pro Glu Gln Gln Glu Ile His 930935 940 Gln Ser Glu Glu Ile Pro Gly Val Leu Thr Asn Thr Ile Ser Gln Leu945 950 955 960 Gly Asn Gln Ile Arg His Asn Phe Asp Leu Tyr Val Tyr LysLys Asp 965 970 975 Gln Pro Gln Ile His Ser Ser Lys Pro Val Arg Val IleIle Ile Glu 980 985 990 Ser Ser Glu Ser Leu Phe Ala Leu Lys 995 1000 112871 DNA Mycoplasma hyopneumoniae 11 atgaaaaaaa acaagctaaa atatttaattttctcaatta ttggaattag tacaattata 60 agtcttgctg ttacaattcc ttatgcactttcatcccaag ccgaaaaata taatctagaa 120 ctaaattctt ataacattga tcttggaaaagcacaaaatt tgaactcaag aactaatttt 180 aatagtgctg aatttgataa attagttgcaaatttaaagg taaaacctaa atttgccaag 240 cgactaaacg cttttgatgc tctaaattttcactttgata aatcttatag tttcgatcta 300 gctgatgcag ttgatttaag tagtctaagtcaaaaatatc ctgatctaag ttttaaattg 360 gttatccctg ataataaatc caggtttgaaatcaaagaaa ataagctaaa aaatatcgga 420 cttaatgtaa ctaacacttc aaaaaccataaattatacag caaaattcga ccttgatttc 480 tcaggtcaag aaaagtcttt ccaatttctacccgaaaatt tcactggcca aattagtctt 540 agaaatcttg aatcacttaa aggaaaaaccgcaactgaaa tagcaatttt attttataat 600 gcttgactaa aacggtttaa taaactttctgattcaaaaa ttgccttata tgaaactttt 660 ggcgaatttg gtggggcttc ctttagcctaaattctgaac caatttttat ccttccagaa 720 aattttgaaa tcaaaccgga tctaaaagataataaactag tttttgcaag tataaatgat 780 gaaaaaaatg agcttgttct taatatggttttatatgata aaacagctaa aactgagaaa 840 atttttcccc ttagatttgt tgatctcccaaaaacaaatc agaaatatgg ggaaaaattt 900 ttagcaagtt ttttgaaaaa ctatgaatttaatagtgaaa tttcaaaata tctagccaaa 960 aataacttag atattgcaca attattttcattaccttctg atccaaaaag tcttgattta 1020 actaaatttg agtcctgatt tattcaaaaatcagtgccaa atacaacttt ttttgctgat 1080 attaaaggtt taattcctaa ttttgagaccaaaaaagcag cttttttagt taaaaaacct 1140 gaaaaagttg gtcagaataa gaatttattaactattaatt taaaattaga aggaactttt 1200 ttagtaaatg atcaagttcc tgcaggtctaaatttgactc aggataaaca ctatacttat 1260 aatttcgact ttgactacga tgcaacacaagaaatttatt ctggatattt tcgaaatgcg 1320 cttgaattat ttgatgctag aacggcaaaaaatcttgata atttaaaact tgaggtcaaa 1380 aacgatcttc cagtaacggt tttcgcctcaacaattaata caaaaattgc ccatctttta 1440 aataaacccc ttgaacttaa gggaattactaaaaaaatga gtcctttatt tgattttctt 1500 aatttttcaa caagtaaaaa tgaaaaattagaaacaaaaa tggctccacc aaatgctaag 1560 atgcaaaatg ttggtgcaat tttatttaatgaagaggtaa aacaacaaga aagtcaggta 1620 aaggatcagg caaaacaaga aaaatcaagtaaagattccc aaagtaaaca aactgatcaa 1680 agtgaaaaag aaccaaaagt tgaaactaaaacaatccagg cagaaaatgg aggaacttat 1740 ttatctaaac tttttgaaaa tttagaaaaaactagtttcc caacaaacac tctattatat 1800 ttatcaactt tttatcggga taaatttattttaaaattag aactaaaagc tgaaggaata 1860 acaaaagaaa cacttgagat taaaattgacaaagttgctc ctgataataa agcttatcaa 1920 gcattagtcc aaagtacaaa tacggatttattccttgatt gacgatcaaa tataaccaca 1980 acaacagaaa aataccaaaa taaaccagtaattgcatcga ttagcgcact aaataatccg 2040 aatttaaaat ttaaggtaaa tccagaaccttcaaataaat cgcagcaaaa agtacatcta 2100 gatcaagccg gtatttattt agccgaagggggaataagtc ttgaaaactt aagtcaagaa 2160 caagcaaaaa atcttaaact tgatgaaggcaagacaattt tttatgcctt taaacccact 2220 aaattatcac gaagatcact tttaagatattttctattaa gcgcaagtga taattctagt 2280 tcaaaattca gtttattaat cgaaccagaaatattactaa ccgggtttaa taaaattggt 2340 gctgattttg aaaaggtaga gcaaaataataaaaatcaat taaaatggac cgatgcctca 2400 ggtgggctgc aaaaaacttt taacgggacttatcaagata tttattattt ccttttacaa 2460 cttctccaac ataataaagt tgcgctttatcctaaaaatc aatcagataa atcacatgat 2520 ttcctcaacg ctccggctgc tacaatggttctagtggcaa cagttgaaag cgaaaataca 2580 gaaaaatacc ttaaaatgaa gcttttttcaagtgattatc aaaatgggaa aaaggaaatt 2640 tttacctgaa aaaccaaaat tgagagccaatttcaaaatc tcgatctagc taaaaatcta 2700 actttaggta caacaaaaag caataatcaagaaaatattg acaaagaaca acaagatgat 2760 agtagaaaac cgaccggaat aacactaaaaggttttgccc tctttgataa accaaaagat 2820 aatcaaaaat ataataatat ccttgaaaaattccttagcg aatatatgga a 2871 12 957 PRT Mycoplasma hyopneumoniae 12 MetLys Lys Asn Lys Leu Lys Tyr Leu Ile Phe Ser Ile Ile Gly Ile 1 5 10 15Ser Thr Ile Ile Ser Leu Ala Val Thr Ile Pro Tyr Ala Leu Ser Ser 20 25 30Gln Ala Glu Lys Tyr Asn Leu Glu Leu Asn Ser Tyr Asn Ile Asp Leu 35 40 45Gly Lys Ala Gln Asn Leu Asn Ser Arg Thr Asn Phe Asn Ser Ala Glu 50 55 60Phe Asp Lys Leu Val Ala Asn Leu Lys Val Lys Pro Lys Phe Ala Lys 65 70 7580 Arg Leu Asn Ala Phe Asp Ala Leu Asn Phe His Phe Asp Lys Ser Tyr 85 9095 Ser Phe Asp Leu Ala Asp Ala Val Asp Leu Ser Ser Leu Ser Gln Lys 100105 110 Tyr Pro Asp Leu Ser Phe Lys Leu Val Ile Pro Asp Asn Lys Ser Arg115 120 125 Phe Glu Ile Lys Glu Asn Lys Leu Lys Asn Ile Gly Leu Asn ValThr 130 135 140 Asn Thr Ser Lys Thr Ile Asn Tyr Thr Ala Lys Phe Asp LeuAsp Phe 145 150 155 160 Ser Gly Gln Glu Lys Ser Phe Gln Phe Leu Pro GluAsn Phe Thr Gly 165 170 175 Gln Ile Ser Leu Arg Asn Leu Glu Ser Leu LysGly Lys Thr Ala Thr 180 185 190 Glu Ile Ala Ile Leu Phe Tyr Asn Ala TrpLeu Lys Arg Phe Asn Lys 195 200 205 Leu Ser Asp Ser Lys Ile Ala Leu TyrGlu Thr Phe Gly Glu Phe Gly 210 215 220 Gly Ala Ser Phe Ser Leu Asn SerGlu Pro Ile Phe Ile Leu Pro Glu 225 230 235 240 Asn Phe Glu Ile Lys ProAsp Leu Lys Asp Asn Lys Leu Val Phe Ala 245 250 255 Ser Ile Asn Asp GluLys Asn Glu Leu Val Leu Asn Met Val Leu Tyr 260 265 270 Asp Lys Thr AlaLys Thr Glu Lys Ile Phe Pro Leu Arg Phe Val Asp 275 280 285 Leu Pro LysThr Asn Gln Lys Tyr Gly Glu Lys Phe Leu Ala Ser Phe 290 295 300 Leu LysAsn Tyr Glu Phe Asn Ser Glu Ile Ser Lys Tyr Leu Ala Lys 305 310 315 320Asn Asn Leu Asp Ile Ala Gln Leu Phe Ser Leu Pro Ser Asp Pro Lys 325 330335 Ser Leu Asp Leu Thr Lys Phe Glu Ser Trp Phe Ile Gln Lys Ser Val 340345 350 Pro Asn Thr Thr Phe Phe Ala Asp Ile Lys Gly Leu Ile Pro Asn Phe355 360 365 Glu Thr Lys Lys Ala Ala Phe Leu Val Lys Lys Pro Glu Lys ValGly 370 375 380 Gln Asn Lys Asn Leu Leu Thr Ile Asn Leu Lys Leu Glu GlyThr Phe 385 390 395 400 Leu Val Asn Asp Gln Val Pro Ala Gly Leu Asn LeuThr Gln Asp Lys 405 410 415 His Tyr Thr Tyr Asn Phe Asp Phe Asp Tyr AspAla Thr Gln Glu Ile 420 425 430 Tyr Ser Gly Tyr Phe Arg Asn Ala Leu GluLeu Phe Asp Ala Arg Thr 435 440 445 Ala Lys Asn Leu Asp Asn Leu Lys LeuGlu Val Lys Asn Asp Leu Pro 450 455 460 Val Thr Val Phe Ala Ser Thr IleAsn Thr Lys Ile Ala His Leu Leu 465 470 475 480 Asn Lys Pro Leu Glu LeuLys Gly Ile Thr Lys Lys Met Ser Pro Leu 485 490 495 Phe Asp Phe Leu AsnPhe Ser Thr Ser Lys Asn Glu Lys Leu Glu Thr 500 505 510 Lys Met Ala ProPro Asn Ala Lys Met Gln Asn Val Gly Ala Ile Leu 515 520 525 Phe Asn GluGlu Val Lys Gln Gln Glu Ser Gln Val Lys Asp Gln Ala 530 535 540 Lys GlnGlu Lys Ser Ser Lys Asp Ser Gln Ser Lys Gln Thr Asp Gln 545 550 555 560Ser Glu Lys Glu Pro Lys Val Glu Thr Lys Thr Ile Gln Ala Glu Asn 565 570575 Gly Gly Thr Tyr Leu Ser Lys Leu Phe Glu Asn Leu Glu Lys Thr Ser 580585 590 Phe Pro Thr Asn Thr Leu Leu Tyr Leu Ser Thr Phe Tyr Arg Asp Lys595 600 605 Phe Ile Leu Lys Leu Glu Leu Lys Ala Glu Gly Ile Thr Lys GluThr 610 615 620 Leu Glu Ile Lys Ile Asp Lys Val Ala Pro Asp Asn Lys AlaTyr Gln 625 630 635 640 Ala Leu Val Gln Ser Thr Asn Thr Asp Leu Phe LeuAsp Trp Arg Ser 645 650 655 Asn Ile Thr Thr Thr Thr Glu Lys Tyr Gln AsnLys Pro Val Ile Ala 660 665 670 Ser Ile Ser Ala Leu Asn Asn Pro Asn LeuLys Phe Lys Val Asn Pro 675 680 685 Glu Pro Ser Asn Lys Ser Gln Gln LysVal His Leu Asp Gln Ala Gly 690 695 700 Ile Tyr Leu Ala Glu Gly Gly IleSer Leu Glu Asn Leu Ser Gln Glu 705 710 715 720 Gln Ala Lys Asn Leu LysLeu Asp Glu Gly Lys Thr Ile Phe Tyr Ala 725 730 735 Phe Lys Pro Thr LysLeu Ser Arg Arg Ser Leu Leu Arg Tyr Phe Leu 740 745 750 Leu Ser Ala SerAsp Asn Ser Ser Ser Lys Phe Ser Leu Leu Ile Glu 755 760 765 Pro Glu IleLeu Leu Thr Gly Phe Asn Lys Ile Gly Ala Asp Phe Glu 770 775 780 Lys ValGlu Gln Asn Asn Lys Asn Gln Leu Lys Trp Thr Asp Ala Ser 785 790 795 800Gly Gly Leu Gln Lys Thr Phe Asn Gly Thr Tyr Gln Asp Ile Tyr Tyr 805 810815 Phe Leu Leu Gln Leu Leu Gln His Asn Lys Val Ala Leu Tyr Pro Lys 820825 830 Asn Gln Ser Asp Lys Ser His Asp Phe Leu Asn Ala Pro Ala Ala Thr835 840 845 Met Val Leu Val Ala Thr Val Glu Ser Glu Asn Thr Glu Lys TyrLeu 850 855 860 Lys Met Lys Leu Phe Ser Ser Asp Tyr Gln Asn Gly Lys LysGlu Ile 865 870 875 880 Phe Thr Trp Lys Thr Lys Ile Glu Ser Gln Phe GlnAsn Leu Asp Leu 885 890 895 Ala Lys Asn Leu Thr Leu Gly Thr Thr Lys SerAsn Asn Gln Glu Asn 900 905 910 Ile Asp Lys Glu Gln Gln Asp Asp Ser ArgLys Pro Thr Gly Ile Thr 915 920 925 Leu Lys Gly Phe Ala Leu Phe Asp LysPro Lys Asp Asn Gln Lys Tyr 930 935 940 Asn Asn Ile Leu Glu Lys Phe LeuSer Glu Tyr Met Glu 945 950 955 13 2835 DNA Mycoplasma hyopneumoniae 13atgaagttag caaaattact taaaaaacct ttttgattaa taacaacaat tgccggaatt 60agtcttagtt tatcagccgc tgttggtata gttgtcggaa ttaattctta taataaatca 120tattattctt atctaaatga aaatccaagt cagctaaaaa ctactaaaac aacaaaaata 180tcccagcaag attttgataa aatagtctca aatttaaaaa ttagggataa ttttaagaaa 240atatcagcaa aaacagcttt atcagcggta aaaaatgatt tataccggta tgacttagtt 300cgggcttttg aattttcaag tttagaaact aacaactatc aaattagttt tgatttagaa 360aatgcagtag ttgatcaaaa ttcaattaaa aatgtgctag tttttgcaaa atctgaaaaa 420gatcaagtaa catattcaaa acaaattgaa cttaaagggt ttgctcaaga tgatgaagct 480gcaggcgatc ttgttaaatt ccaaattgat caaagaaaat cctttgttaa tctttataaa 540tttgattatt ctttttctga atttcaaaga attcttagcg aaaattatcg acaaattaga 600aatacaaatt cttttacaag gttggcaaat gctttgattt cctcaaaagc gagtctttca 660ctttataatt ccttagggca accagtattt ttagatgaaa attatcgctt agaaccagtt 720ttgaattcaa aaaaagaatt aaatttacta gaaaaaaata agaaattgta tttagaactt 780aatttagttg aaaaagagag ccaaaagaaa attaatttaa cactagaaat ccgtccatta 840ttaacaaatc aagaatttac tagtgagtta aaaactttat ttgaatcaaa tttagaccaa 900aatcttagcc taaatcttga actaaaaaat gctcttttcc atgatagaac cagtttttct 960gagtatttat atggaagtcc acagcaaaga actaaaactg atgaagtaaa acagaaagct 1020aaggaattaa aggatctttt tggttttaga tcagcaaaat tctgacagga tacaaaattt 1080ggaacttttt atgtaataat taagccccaa cttttagatc ctgcaaaaat tagtcaagaa 1140gataagaaaa aacttttagc tgataaaaaa atccgttttg aagttctaac taccttaaaa 1200agaaaagcgc ttgatcaaca agatgttctc actgatcttc cagttttagt cgatctaagc 1260cttgattcta ataaatacga aacagccata agtcaaattt ttaattcaac aaagacaacc 1320aaagaattta aaatgcaaga atatgaagat agagcgaagt tatcaaccaa agaaatcaaa 1380gaaacaattg ataaattagc aaatcttgcc gcaaaagtta gtaatttatc cgaaccaagt 1440gatgaagttg ttcgtgctgt ctatttatta aatacaggga aatatctttt tgatgatgag 1500atccagcaag aaaaaactaa tcttaaaaaa ataatagaac aagcccgaat gaaagctgac 1560accaagaatt tggctccaaa agtacctagt cctattcaaa aaccaactac atctgcaact 1620tctagtggaa ctactaagac atcaacaggg acagaaaaaa aagtttcagt aagtgctttt 1680tctgatataa ttagtatgaa aaaccaacct gaacaaacaa ctaagaacgg tcaggtccaa 1740gcttcttcta caagtcagag tccaaaatca agtcttagcc aaaacagcgg acaaaattca 1800ataactttag aagaaaaatt tggacataca atttgaaagt tactaaatac atcacaaatt 1860tataattttg aaaacaccca agggcaatat acaatctcaa tagaggatga taaattagtt 1920tttgacttta agcttgtatc aaaagcagat cgagcaatta tttatcaagg atctaaaatt 1980agtcttggtg gtctaattaa ttctgataag tctgcctatg atgagattaa acaatttagc 2040ccagatcttt tccttgatgc aacaatagga gaacaatctg attataaaaa caagcaaaaa 2100aaagattata ctttaaaatc gttaagagat ttaatgggta atggctttgt ttataaacca 2160gaaactaaat cgaatccaca agaaaatgta ctaaaattac aaacaggatc agagcaaaaa 2220aaacctctac cagggcttag atcaggatta atttatattg catttaccgt taataatatc 2280aataaaaatg attataaacc tcattatcta ataagagata aaaatgataa aggtgtcttc 2340attcagagat atcaagataa ggaagaacca aacgcttttg agattagaat tgattcatat 2400gagcctgatg acttcaggga taaacaattt caggctgctg atacgatatt agatgcaagt 2460ggttcaattg atcctcgatc aaagaaaaaa attattctcc gtcaaaacgc tgattattta 2520ttagtagttt ataagtcaaa aaaagatatt gtaacagagc tttattcact accttcagca 2580caagataata acaaagaaaa gattgttaaa ataaaaaata gaaaatcatt tccctctcaa 2640ggttatacag ttcaaggttc attattatat tctttattta gtcctaataa aattggagat 2700agtcagaagc cagcccaaca accgccagct gtaagtataa aagcaatagc attatttgat 2760aaaaaatcat ttacaaacga tacagaaaaa atgcgtttaa taaataatgc ttttattagt 2820aattatataa aacaa 2835 14 945 PRT Mycoplasma hyopneumoniae 14 Met Lys LeuAla Lys Leu Leu Lys Lys Pro Phe Trp Leu Ile Thr Thr 1 5 10 15 Ile AlaGly Ile Ser Leu Ser Leu Ser Ala Ala Val Gly Ile Val Val 20 25 30 Gly IleAsn Ser Tyr Asn Lys Ser Tyr Tyr Ser Tyr Leu Asn Glu Asn 35 40 45 Pro SerGln Leu Lys Thr Thr Lys Thr Thr Lys Ile Ser Gln Gln Asp 50 55 60 Phe AspLys Ile Val Ser Asn Leu Lys Ile Arg Asp Asn Phe Lys Lys 65 70 75 80 IleSer Ala Lys Thr Ala Leu Ser Ala Val Lys Asn Asp Leu Tyr Arg 85 90 95 TyrAsp Leu Val Arg Ala Phe Glu Phe Ser Ser Leu Glu Thr Asn Asn 100 105 110Tyr Gln Ile Ser Phe Asp Leu Glu Asn Ala Val Val Asp Gln Asn Ser 115 120125 Ile Lys Asn Val Leu Val Phe Ala Lys Ser Glu Lys Asp Gln Val Thr 130135 140 Tyr Ser Lys Gln Ile Glu Leu Lys Gly Phe Ala Gln Asp Asp Glu Ala145 150 155 160 Ala Gly Asp Leu Val Lys Phe Gln Ile Asp Gln Arg Lys SerPhe Val 165 170 175 Asn Leu Tyr Lys Phe Asp Tyr Ser Phe Ser Glu Phe GlnArg Ile Leu 180 185 190 Ser Glu Asn Tyr Arg Gln Ile Arg Asn Thr Asn SerPhe Thr Arg Leu 195 200 205 Ala Asn Ala Leu Ile Ser Ser Lys Ala Ser LeuSer Leu Tyr Asn Ser 210 215 220 Leu Gly Gln Pro Val Phe Leu Asp Glu AsnTyr Arg Leu Glu Pro Val 225 230 235 240 Leu Asn Ser Lys Lys Glu Leu AsnLeu Leu Glu Lys Asn Lys Lys Leu 245 250 255 Tyr Leu Glu Leu Asn Leu ValGlu Lys Glu Ser Gln Lys Lys Ile Asn 260 265 270 Leu Thr Leu Glu Ile ArgPro Leu Leu Thr Asn Gln Glu Phe Thr Ser 275 280 285 Glu Leu Lys Thr LeuPhe Glu Ser Asn Leu Asp Gln Asn Leu Ser Leu 290 295 300 Asn Leu Glu LeuLys Asn Ala Leu Phe His Asp Arg Thr Ser Phe Ser 305 310 315 320 Glu TyrLeu Tyr Gly Ser Pro Gln Gln Arg Thr Lys Thr Asp Glu Val 325 330 335 LysGln Lys Ala Lys Glu Leu Lys Asp Leu Phe Gly Phe Arg Ser Ala 340 345 350Lys Phe Trp Gln Asp Thr Lys Phe Gly Thr Phe Tyr Val Ile Ile Lys 355 360365 Pro Gln Leu Leu Asp Pro Ala Lys Ile Ser Gln Glu Asp Lys Lys Lys 370375 380 Leu Leu Ala Asp Lys Lys Ile Arg Phe Glu Val Leu Thr Thr Leu Lys385 390 395 400 Arg Lys Ala Leu Asp Gln Gln Asp Val Leu Thr Asp Leu ProVal Leu 405 410 415 Val Asp Leu Ser Leu Asp Ser Asn Lys Tyr Glu Thr AlaIle Ser Gln 420 425 430 Ile Phe Asn Ser Thr Lys Thr Thr Lys Glu Phe LysMet Gln Glu Tyr 435 440 445 Glu Asp Arg Ala Lys Leu Ser Thr Lys Glu IleLys Glu Thr Ile Asp 450 455 460 Lys Leu Ala Asn Leu Ala Ala Lys Val SerAsn Leu Ser Glu Pro Ser 465 470 475 480 Asp Glu Val Val Arg Ala Val TyrLeu Leu Asn Thr Gly Lys Tyr Leu 485 490 495 Phe Asp Asp Glu Ile Gln GlnGlu Lys Thr Asn Leu Lys Lys Ile Ile 500 505 510 Glu Gln Ala Arg Met LysAla Asp Thr Lys Asn Leu Ala Pro Lys Val 515 520 525 Pro Ser Pro Ile GlnLys Pro Thr Thr Ser Ala Thr Ser Ser Gly Thr 530 535 540 Thr Lys Thr SerThr Gly Thr Glu Lys Lys Val Ser Val Ser Ala Phe 545 550 555 560 Ser AspIle Ile Ser Met Lys Asn Gln Pro Glu Gln Thr Thr Lys Asn 565 570 575 GlyGln Val Gln Ala Ser Ser Thr Ser Gln Ser Pro Lys Ser Ser Leu 580 585 590Ser Gln Asn Ser Gly Gln Asn Ser Ile Thr Leu Glu Glu Lys Phe Gly 595 600605 His Thr Ile Trp Lys Leu Leu Asn Thr Ser Gln Ile Tyr Asn Phe Glu 610615 620 Asn Thr Gln Gly Gln Tyr Thr Ile Ser Ile Glu Asp Asp Lys Leu Val625 630 635 640 Phe Asp Phe Lys Leu Val Ser Lys Ala Asp Arg Ala Ile IleTyr Gln 645 650 655 Gly Ser Lys Ile Ser Leu Gly Gly Leu Ile Asn Ser AspLys Ser Ala 660 665 670 Tyr Asp Glu Ile Lys Gln Phe Ser Pro Asp Leu PheLeu Asp Ala Thr 675 680 685 Ile Gly Glu Gln Ser Asp Tyr Lys Asn Lys GlnLys Lys Asp Tyr Thr 690 695 700 Leu Lys Ser Leu Arg Asp Leu Met Gly AsnGly Phe Val Tyr Lys Pro 705 710 715 720 Glu Thr Lys Ser Asn Pro Gln GluAsn Val Leu Lys Leu Gln Thr Gly 725 730 735 Ser Glu Gln Lys Lys Pro LeuPro Gly Leu Arg Ser Gly Leu Ile Tyr 740 745 750 Ile Ala Phe Thr Val AsnAsn Ile Asn Lys Asn Asp Tyr Lys Pro His 755 760 765 Tyr Leu Ile Arg AspLys Asn Asp Lys Gly Val Phe Ile Gln Arg Tyr 770 775 780 Gln Asp Lys GluGlu Pro Asn Ala Phe Glu Ile Arg Ile Asp Ser Tyr 785 790 795 800 Glu ProAsp Asp Phe Arg Asp Lys Gln Phe Gln Ala Ala Asp Thr Ile 805 810 815 LeuAsp Ala Ser Gly Ser Ile Asp Pro Arg Ser Lys Lys Lys Ile Ile 820 825 830Leu Arg Gln Asn Ala Asp Tyr Leu Leu Val Val Tyr Lys Ser Lys Lys 835 840845 Asp Ile Val Thr Glu Leu Tyr Ser Leu Pro Ser Ala Gln Asp Asn Asn 850855 860 Lys Glu Lys Ile Val Lys Ile Lys Asn Arg Lys Ser Phe Pro Ser Gln865 870 875 880 Gly Tyr Thr Val Gln Gly Ser Leu Leu Tyr Ser Leu Phe SerPro Asn 885 890 895 Lys Ile Gly Asp Ser Gln Lys Pro Ala Gln Gln Pro ProAla Val Ser 900 905 910 Ile Lys Ala Ile Ala Leu Phe Asp Lys Lys Ser PheThr Asn Asp Thr 915 920 925 Glu Lys Met Arg Leu Ile Asn Asn Ala Phe IleSer Asn Tyr Ile Lys 930 935 940 Gln 945 15 1380 DNA Mycoplasmahyopneumoniae 15 gtgattgagg gcttaaaatc aaaggcaaat actcaaaaaa cagaaaaaaatagccccaca 60 caaccgaaaa aaccagaggt ttcactagct aaaacaacag aaaattcagcaaaaacagtc 120 aaggtaagca cttttgcaga agaagctaag ggtcaaagtc aaagtcagcaaacacaacca 180 gtttccactt catcgcctca aactagtcaa aattcagttt ctaattccacaagcagtacg 240 aatttagcct tagaaaatga aaaatttggg acaagcattt gaacagcttttaatttcgct 300 aatatttata atcttgaaaa tacaaaaagc gaatatgaga tctcaactttaggaaataag 360 ctattttttg attttaaatt agttgataaa actaatcaaa atctaattttggctcagtcc 420 aaaattagtc ttaataatat tattaattct aataaatctg cctatgatataattaagaaa 480 ttcaatcccg atgtatttct agatggaaca attaattatc aagatcaaggaaaagataaa 540 aaagaattta tcctaaaaga tttaagtgat aataaattaa tatttaaatcagaagatgca 600 attcaaactg atcaaggttt agagctaaag aaacctttga aattaagcccgacaacgaac 660 tcttcttcta ctacttcaca aaagactaat aaaaaggatg atattggagtgttttgacta 720 gcgcttcaag ttaataatat aacagatttc aaaaatcatc atctaatatccgatggaaaa 780 ggaaatggaa taattcttaa caaatacaag gtcaaggatg aaactggttatcaattagga 840 ctagaatatc ctggaaggaa tgaaaataat tttattactg atattgttgatctagtcgac 900 ggttttatca aatttatttt tggatgaaaa caagaccaaa ataatagtagttttttggac 960 acaccctcac ttttaattga ttttaacaag tataaaaaca aaaaaaatactgaatttatc 1020 aaggcgaata caaaaattct tttagaggtt gtagaaaaca atgatcgactttctgtttca 1080 gtattttctt ctcaagcagg aaaaaatcat aaacaaatta tagaaaatagaatgcataga 1140 agtttacatt ataaaaaagc agacaaagcc aaagaaggtg taagcccaatcccaagtttt 1200 actgatattt taaatgaatt acaaattgga gctactgata gcgatccaaaaactcaaaag 1260 gcaccagtaa cattcaaagc gtttatgatg tcaaatgata aaaatctagtatttggatca 1320 aacattaata atcaagaaat tcgccaagcg cttattgacg cttatatagttgataagaat 1380 16 460 PRT Mycoplasma hyopneumoniae 16 Val Ile Glu GlyLeu Lys Ser Lys Ala Asn Thr Gln Lys Thr Glu Lys 1 5 10 15 Asn Ser ProThr Gln Pro Lys Lys Pro Glu Val Ser Leu Ala Lys Thr 20 25 30 Thr Glu AsnSer Ala Lys Thr Val Lys Val Ser Thr Phe Ala Glu Glu 35 40 45 Ala Lys GlyGln Ser Gln Ser Gln Gln Thr Gln Pro Val Ser Thr Ser 50 55 60 Ser Pro GlnThr Ser Gln Asn Ser Val Ser Asn Ser Thr Ser Ser Thr 65 70 75 80 Asn LeuAla Leu Glu Asn Glu Lys Phe Gly Thr Ser Ile Trp Thr Ala 85 90 95 Phe AsnPhe Ala Asn Ile Tyr Asn Leu Glu Asn Thr Lys Ser Glu Tyr 100 105 110 GluIle Ser Thr Leu Gly Asn Lys Leu Phe Phe Asp Phe Lys Leu Val 115 120 125Asp Lys Thr Asn Gln Asn Leu Ile Leu Ala Gln Ser Lys Ile Ser Leu 130 135140 Asn Asn Ile Ile Asn Ser Asn Lys Ser Ala Tyr Asp Ile Ile Lys Lys 145150 155 160 Phe Asn Pro Asp Val Phe Leu Asp Gly Thr Ile Asn Tyr Gln AspGln 165 170 175 Gly Lys Asp Lys Lys Glu Phe Ile Leu Lys Asp Leu Ser AspAsn Lys 180 185 190 Leu Ile Phe Lys Ser Glu Asp Ala Ile Gln Thr Asp GlnGly Leu Glu 195 200 205 Leu Lys Lys Pro Leu Lys Leu Ser Pro Thr Thr AsnSer Ser Ser Thr 210 215 220 Thr Ser Gln Lys Thr Asn Lys Lys Asp Asp IleGly Val Phe Trp Leu 225 230 235 240 Ala Leu Gln Val Asn Asn Ile Thr AspPhe Lys Asn His His Leu Ile 245 250 255 Ser Asp Gly Lys Gly Asn Gly IleIle Leu Asn Lys Tyr Lys Val Lys 260 265 270 Asp Glu Thr Gly Tyr Gln LeuGly Leu Glu Tyr Pro Gly Arg Asn Glu 275 280 285 Asn Asn Phe Ile Thr AspIle Val Asp Leu Val Asp Gly Phe Ile Lys 290 295 300 Phe Ile Phe Gly TrpLys Gln Asp Gln Asn Asn Ser Ser Phe Leu Asp 305 310 315 320 Thr Pro SerLeu Leu Ile Asp Phe Asn Lys Tyr Lys Asn Lys Lys Asn 325 330 335 Thr GluPhe Ile Lys Ala Asn Thr Lys Ile Leu Leu Glu Val Val Glu 340 345 350 AsnAsn Asp Arg Leu Ser Val Ser Val Phe Ser Ser Gln Ala Gly Lys 355 360 365Asn His Lys Gln Ile Ile Glu Asn Arg Met His Arg Ser Leu His Tyr 370 375380 Lys Lys Ala Asp Lys Ala Lys Glu Gly Val Ser Pro Ile Pro Ser Phe 385390 395 400 Thr Asp Ile Leu Asn Glu Leu Gln Ile Gly Ala Thr Asp Ser AspPro 405 410 415 Lys Thr Gln Lys Ala Pro Val Thr Phe Lys Ala Phe Met MetSer Asn 420 425 430 Asp Lys Asn Leu Val Phe Gly Ser Asn Ile Asn Asn GlnGlu Ile Arg 435 440 445 Gln Ala Leu Ile Asp Ala Tyr Ile Val Asp Lys Asn450 455 460 17 1353 DNA Mycoplasma hyopneumoniae 17 atgaagttagcaaaattact taaaaaacct ttttgattaa taacaacaat tgccggaatt 60 agtcttagtttatcagccgc tgttggtaca gttgtcggaa ttaattctta taataaatca 120 tattattcttatctaaatca gatcccgagt cagctaaaag tagcaaaaaa tgctaaaatt 180 agtcaggaaaaatttgattc aattgtttta aatcttaaaa ttaaagataa ttttaaaaaa 240 tgatcggcaaaaacagtttt aactgctgcc aaaagtgatc tttatcgtta taatcttgtt 300 tctgcttttgatttaagtga actaataaac aatgattatt tagtaagttt tgatcttgaa 360 aatgcagtagttgatcaaaa ttcaattaaa aatgttgtta tttatgcaaa atctgataag 420 gatcaaataacttattcaaa acaaattgta cttaaaggct ttggaaatac agaacaagcg 480 agaactaattttgattttag ccaaattgat tcaagcaagt cttttgttga tctttcaagg 540 gcaaatctaactttgacgga attccaaatt ttacttgccc aaaattttga aaatgaaaga 600 ggaagtaattgattttcacg acttgaaaga gctttggttg catcaaaagc gagtctttca 660 ctttataattccttaggaga acccgtattt ttaggcccag attatcaatt agacccagtt 720 ttggaccgaaaaaaattatt aactttgtta aataaagatg gaaaattagt tcttggactt 780 aatttagtgcaaatttcaac taaaaaaact atgaatttaa atcttgaagt tcgcggcgcg 840 atttcaaatcaggaaatttc taaaattcta aaatcctgac ttgaaacaaa tcttcaaggc 900 aaattaaaaaccaaagatga tttgcaaatg gcactagtaa aagataaaat tagcctctct 960 gattattgatatggatctcc gaattcaaaa gtaaatacat cccaaatttt aacaaaaagt 1020 aaagaatttaaagatctttt tgatttaagt gagacaaatt tttttcttaa taccaaaatc 1080 ggaactgtctatttaagtat tattcccaaa cttttagatc caagtcagat ttctgttgtt 1140 gataagaaaaaactagttga aaatcaaaaa attcgctttg aaattactgc ttctttaaaa 1200 cgaaaagctattgataaaaa atttatcatc caggatcttc cagtttttgt tgatctaaaa 1260 gttgattttaataaatacca agccgctgtt gcccaaatgt ttggaacgat aaaagcagtt 1320 aaagaattttcaatgcctga agatcaagat gca 1353 18 451 PRT Mycoplasma hyopneumoniae 18Met Lys Leu Ala Lys Leu Leu Lys Lys Pro Phe Trp Leu Ile Thr Thr 1 5 1015 Ile Ala Gly Ile Ser Leu Ser Leu Ser Ala Ala Val Gly Thr Val Val 20 2530 Gly Ile Asn Ser Tyr Asn Lys Ser Tyr Tyr Ser Tyr Leu Asn Gln Ile 35 4045 Pro Ser Gln Leu Lys Val Ala Lys Asn Ala Lys Ile Ser Gln Glu Lys 50 5560 Phe Asp Ser Ile Val Leu Asn Leu Lys Ile Lys Asp Asn Phe Lys Lys 65 7075 80 Trp Ser Ala Lys Thr Val Leu Thr Ala Ala Lys Ser Asp Leu Tyr Arg 8590 95 Tyr Asn Leu Val Ser Ala Phe Asp Leu Ser Glu Leu Ile Asn Asn Asp100 105 110 Tyr Leu Val Ser Phe Asp Leu Glu Asn Ala Val Val Asp Gln AsnSer 115 120 125 Ile Lys Asn Val Val Ile Tyr Ala Lys Ser Asp Lys Asp GlnIle Thr 130 135 140 Tyr Ser Lys Gln Ile Val Leu Lys Gly Phe Gly Asn ThrGlu Gln Ala 145 150 155 160 Arg Thr Asn Phe Asp Phe Ser Gln Ile Asp SerSer Lys Ser Phe Val 165 170 175 Asp Leu Ser Arg Ala Asn Leu Thr Leu ThrGlu Phe Gln Ile Leu Leu 180 185 190 Ala Gln Asn Phe Glu Asn Glu Arg GlySer Asn Trp Phe Ser Arg Leu 195 200 205 Glu Arg Ala Leu Val Ala Ser LysAla Ser Leu Ser Leu Tyr Asn Ser 210 215 220 Leu Gly Glu Pro Val Phe LeuGly Pro Asp Tyr Gln Leu Asp Pro Val 225 230 235 240 Leu Asp Arg Lys LysLeu Leu Thr Leu Leu Asn Lys Asp Gly Lys Leu 245 250 255 Val Leu Gly LeuAsn Leu Val Gln Ile Ser Thr Lys Lys Thr Met Asn 260 265 270 Leu Asn LeuGlu Val Arg Gly Ala Ile Ser Asn Gln Glu Ile Ser Lys 275 280 285 Ile LeuLys Ser Trp Leu Glu Thr Asn Leu Gln Gly Lys Leu Lys Thr 290 295 300 LysAsp Asp Leu Gln Met Ala Leu Val Lys Asp Lys Ile Ser Leu Ser 305 310 315320 Asp Tyr Trp Tyr Gly Ser Pro Asn Ser Lys Val Asn Thr Ser Gln Ile 325330 335 Leu Thr Lys Ser Lys Glu Phe Lys Asp Leu Phe Asp Leu Ser Glu Thr340 345 350 Asn Phe Phe Leu Asn Thr Lys Ile Gly Thr Val Tyr Leu Ser IleIle 355 360 365 Pro Lys Leu Leu Asp Pro Ser Gln Ile Ser Val Val Asp LysLys Lys 370 375 380 Leu Val Glu Asn Gln Lys Ile Arg Phe Glu Ile Thr AlaSer Leu Lys 385 390 395 400 Arg Lys Ala Ile Asp Lys Lys Phe Ile Ile GlnAsp Leu Pro Val Phe 405 410 415 Val Asp Leu Lys Val Asp Phe Asn Lys TyrGln Ala Ala Val Ala Gln 420 425 430 Met Phe Gly Thr Ile Lys Ala Val LysGlu Phe Ser Met Pro Glu Asp 435 440 445 Gln Asp Ala 450 19 5637 DNAMycoplasma hyopneumoniae 19 atgaaaaaca aaaaatcaac attactatta gccacagcggcggcaattat tggttcaact 60 gtttttggga cagttgttgg cttggcttca aaagttaaatatcggggtgt aaatccaact 120 caaggagtaa tatctcaatt aggactgatt gattctgttgcatttaaacc ttcgattgca 180 aattttacaa gcgattatca aagtgttaaa aaagcacttttaaatgggaa aacctttgat 240 ccaaaaagtt cagaatttac tgattttgtc tcaaaatttgactttttgac taataatggg 300 agaaccgttt tggagatccc gaaaaaatat caggtggttatctcggaatt tagccccgag 360 gatgataaag aacgttttcg tcttggattt catctaaaagaaaaacttga agatggaaat 420 atagctcaat cagcaactaa atttatttat cttttaccacttgatatgcc caaagcggcc 480 ctgggtcaat attcttatat cgttgataaa aattttaataatttaattat ccatccttta 540 tctaattttt ctgctcaatc aataaagccg cttgcactgacccgttcaag tgattttata 600 gcaaaactta atcagtttaa aaatcaggac gaactttgagtttatcttga aaaattcttt 660 gatcttgaag ctctaaaagc aaatattcgt ttgcagacagccgattttag ttttgaaaaa 720 ggcaatttag ttgatccttt tgtttattct tttattagaaatccgcaaaa tggaaaagaa 780 tgagctagtg atcttaatca agatcaaaaa accgtcagactttatcttcg aaccgaattt 840 agtcctcagg ctaaaaccat tttaaaagac tataaatacaaagatgagac tttcttaagt 900 agtatcgatt taaaagcaag taatggaact agtttatttgctaatgaaaa tgatctaaaa 960 gatcaattag atgttgatct tttagatgtc tctgattattttggaggcca atcagagaca 1020 attactagta attcccaagt taaacctgtc cctgctagtgagagatcttt aaaagatcgg 1080 gttaaattta aaaaagatca gcaaaaacca agaattgagaaatttagttt atatgaatat 1140 gatgctctaa gtttttattc ccaacttcag gaattagtttctaaacctaa ttcaattaaa 1200 gatttagtta atgcaacttt agctcgtaat cttcggttttcattaggaaa atataatttt 1260 ctttttgatg atttagccag tcatcttgat tatacttttttagtttcaaa agcaaaaatt 1320 aaacaaagtt caattacaaa aaaattattc attgaattaccaatcaaaat tagtcttaaa 1380 tcttcaattt taggtgatca agaacctaat attaaaactttattcgaaaa agaagtaact 1440 tttaaattag ataacttccg tgatgttgaa atcgaaaaagcttttggact tttatatcca 1500 ggtgttaatg aagaacttga acaagcccga agagagcaaagagcaagttt ggaaaaagaa 1560 aaagcgaaaa agggtcttaa agaatttagc cagcaaaaagatgagaattt aaaagcaata 1620 aataatcaag atggtcttga agaagatgat aatattactgaaagacttcc tgagaattcc 1680 ccgattcaat atcagcaaga aaaggccggt ttaggttcaagtccggataa accttatatg 1740 ataaaggatg tccaaaatca acgttattat ctagcaaaatcacaaattca agaactaatt 1800 aaggccaaag attataccaa attagccaaa cttttatccaatagacatac ttataatatt 1860 tctttaagat taaaagaaca actttttgaa gtaaatccaagaattccaag ctctagagat 1920 atagaaaatg caaaatttgt tctagataaa accgaaaaaaataaatactg gcagatttat 1980 tcaagtgctt ctcctgcttt ccaaaataaa tgatcactttttggatatta ccgttattta 2040 ttaggtcttg atccaaaaca aacaatccac gaattagtaaaattaggaca aaaagcgggt 2100 cttcaatttg aaggatatga aaatcttcct tctgatttcaatcttgaaga tcttaagaat 2160 attaggatta aaacaccttt atttagtcaa aaagataatttcaaattatc tttacttgat 2220 tttaataatt attatgatgg tgaaattaaa gccccagaatttggtcttcc tttattttta 2280 ccaaaagaat taagaaaaaa tagttcaaat attggtagttctcaaaactc taatagccct 2340 tgagaacaag aaattattag ccaatttaaa gatcaaaatctatctaatca ggatcagtta 2400 gcccagttta gtactaaaat ctgggaaaaa atcattggtgatgaaaacga atttgatcaa 2460 aataacaggc ttcagtataa acttttaaaa gatcttcaagaatcttgaat taacaaaact 2520 cgcgataatc tttattggac ttatctaggt gataaacttaaagttaaacc aaaaaataat 2580 ttagatgcta aatttagaca aatttccaat ttacaagagcttttaactgc tttttatacc 2640 tcagctgctc tttctaataa ctgaaattat tatcaagattcaggggcaaa gtcaactatt 2700 atttttgaag aaatagctga gctagatcca aaagtaaaagaaaaagtagg agctgatgtt 2760 tatcaattaa aattccatta tgcaatcggt tttgatgataatgctggcaa gtttaatcaa 2820 gaagtaattc gttcttcaag tagaacaatt tatcttaaaacctcagggaa atccaaatta 2880 gaagcagata caattgatca acttaatcaa gcagttgaaaatgcaccttt aggtcttcaa 2940 agtttttatc ttgatactga aagatttggg gttttccaaaaattagcaac ttccttagca 3000 gttcaacata aacaaaaaga aaaaccacta cctaaaaaactaaataatga tggctatact 3060 ttaattcatg ataaacttaa aaaaccagta attccccaaattagttcaag tcccgaaaaa 3120 gattgatttg aaggtaaatt aaatcaaaac gggcaaagccaaaatgtaaa tgtctcaact 3180 tttggttcaa taatcgagtc cccttatttt agtactaatttccaagaaga agctgattta 3240 gaccaagaag gacaagatga ttcaaaacaa ggaaataagagcctagataa tcaagaagca 3300 ggtcttttaa aacaaaaact ggcaatttta ttagggaatcaatttatcca atattatcaa 3360 caaaatgata aagaaattga attcgagatt atcaatgttgagaaagtttc agagcttagt 3420 ttccgcgttg aatttaaatt agcaaaaact cttgaagacaacggaaaaac tattcgagtt 3480 ttatcagatg agacaatgtc attaattgtt aatactacaattgaaaaagc accagaaatg 3540 agtgctgctc ccgaagtatt cgatactaaa tgggttgagcaatatgatcc aagaaccccg 3600 cttgcggcta agacaaagtt tgtcttaaaa ttcaaagatcaaataccagt tgatgccagc 3660 ggaaatattt ctgataaatg actagcaagt attcctttggtgattcacca gcaaatgttg 3720 cgtcttagcc cggtagttaa aacaataaga gagcttggtctaaaaactga acaacaacaa 3780 caacaacaac aacaacaaca aaagaaagct gttagaaaagaagaagaact ggaaacctat 3840 aatccaaaag acgagtttaa tattcttaat cctttaacaaaagctcaccg tcttacctta 3900 tcaaatttag taaataatga tccaaattat aaaattgaagatttaaaagt aatcaaaaat 3960 gaagcaggtg atcatcaatt agaattttct ctaagagctaataatatcaa aagattaatg 4020 aatacaccaa ttacttttgc tgattataat ccctttttctattttaatga ggactgaaga 4080 aatatagata aatatttaaa taataaagga aatgtgagttctcaacaaca acaacaacaa 4140 caacaacaac caggcggggg taatcaaggc tcgggtctaatccaaagact taataaaaat 4200 attaagcccg aaacttttac ccccgcactc atagctcttaaacgagataa taatactaat 4260 ctttctaact attctgataa aataataatg atcaaaccaaaatatttggt tgaacgatca 4320 attggtgttc cctgatcaac cggccttgat ggttatattggttcagaaca actcaagggc 4380 ggaacttcct caaacggtca aaagcgattt aagcaagattttattcaggc tttaggtctt 4440 aaaaacactg aatatcatgg taaactaggt ctttcaattagaatttttga tcctggaaat 4500 gaactagcaa aaattaagga tgcttcaaat aaaaaaggggaagaaaaact gttaaaatca 4560 tatgatttat ttaaaaacta tttaaatgaa tatgagaaaaaatcccctaa aattgctaag 4620 ggatgaacaa atattcatcc tgatcaaaaa gaatatccaaatccaaatca aaaactacct 4680 gaaaattatc ttaacctagt tttaaatcaa ccttgaaaggttactttata taattcaagt 4740 gattttatta ctaatttatt tgttgaacct gaaggctcagatcggggatc tggagcaaaa 4800 ttaaaacaag taatccagaa gcaagttaat aataactatgctgactgggg gtctgcatat 4860 ctcacgttct ggtatgataa agatatcatt accaatcagccaaatgttat aactgctaac 4920 attgctgatg tctttattaa agatgtaaag gaacttgaagataatacaaa actaattgct 4980 ccaaatatta ctcaatgatg gccaaatatt agcggctcaaaggagaaatt ttataagcca 5040 acagtgtttt ttggtaattg agaaaatgaa aacagcaatatgaattccca ggggcagacc 5100 cctacctggg agaagatcag agaaggattt gctctccaagcgcttaaatc cagctttgat 5160 caaaaaacaa ggacatttgt ccttacaaca aatgctcctttacctttatg aaaatacgga 5220 ccattaggtt tccaaaatgg gccgaatttc aaaacacaagattgaaggct tgttttccaa 5280 aatgatgata accaaatagc cgcgctaaga gtccaggagcaagatcgccc agaaaaatca 5340 agcgaagata aagacaagca aaaatggatt aaatttaaagttgttatccc tgaagaaatg 5400 tttaattccg gtaatatacg ttttgttggg gtaatgcagatccaaggtcc taatacttta 5460 tgacttccag tgattaattc ttcggttatc tatgacttctatcgcggaac aggagattct 5520 aacgatgtcg ccaatcttaa tgtagctcct tgacaggttaaaacaatcgc atttacaaat 5580 aacgccttta ataatgtttt caaagagttt aatatctctaaaaaaatagt agaataa 5637 20 1878 PRT Mycoplasma hyopneumoniae 20 Met LysAsn Lys Lys Ser Thr Leu Leu Leu Ala Thr Ala Ala Ala Ile 1 5 10 15 IleGly Ser Thr Val Phe Gly Thr Val Val Gly Leu Ala Ser Lys Val 20 25 30 LysTyr Arg Gly Val Asn Pro Thr Gln Gly Val Ile Ser Gln Leu Gly 35 40 45 LeuIle Asp Ser Val Ala Phe Lys Pro Ser Ile Ala Asn Phe Thr Ser 50 55 60 AspTyr Gln Ser Val Lys Lys Ala Leu Leu Asn Gly Lys Thr Phe Asp 65 70 75 80Pro Lys Ser Ser Glu Phe Thr Asp Phe Val Ser Lys Phe Asp Phe Leu 85 90 95Thr Asn Asn Gly Arg Thr Val Leu Glu Ile Pro Lys Lys Tyr Gln Val 100 105110 Val Ile Ser Glu Phe Ser Pro Glu Asp Asp Lys Glu Arg Phe Arg Leu 115120 125 Gly Phe His Leu Lys Glu Lys Leu Glu Asp Gly Asn Ile Ala Gln Ser130 135 140 Ala Thr Lys Phe Ile Tyr Leu Leu Pro Leu Asp Met Pro Lys AlaAla 145 150 155 160 Leu Gly Gln Tyr Ser Tyr Ile Val Asp Lys Asn Phe AsnAsn Leu Ile 165 170 175 Ile His Pro Leu Ser Asn Phe Ser Ala Gln Ser IleLys Pro Leu Ala 180 185 190 Leu Thr Arg Ser Ser Asp Phe Ile Ala Lys LeuAsn Gln Phe Lys Asn 195 200 205 Gln Asp Glu Leu Trp Val Tyr Leu Glu LysPhe Phe Asp Leu Glu Ala 210 215 220 Leu Lys Ala Asn Ile Arg Leu Gln ThrAla Asp Phe Ser Phe Glu Lys 225 230 235 240 Gly Asn Leu Val Asp Pro PheVal Tyr Ser Phe Ile Arg Asn Pro Gln 245 250 255 Asn Gly Lys Glu Trp AlaSer Asp Leu Asn Gln Asp Gln Lys Thr Val 260 265 270 Arg Leu Tyr Leu ArgThr Glu Phe Ser Pro Gln Ala Lys Thr Ile Leu 275 280 285 Lys Asp Tyr LysTyr Lys Asp Glu Thr Phe Leu Ser Ser Ile Asp Leu 290 295 300 Lys Ala SerAsn Gly Thr Ser Leu Phe Ala Asn Glu Asn Asp Leu Lys 305 310 315 320 AspGln Leu Asp Val Asp Leu Leu Asp Val Ser Asp Tyr Phe Gly Gly 325 330 335Gln Ser Glu Thr Ile Thr Ser Asn Ser Gln Val Lys Pro Val Pro Ala 340 345350 Ser Glu Arg Ser Leu Lys Asp Arg Val Lys Phe Lys Lys Asp Gln Gln 355360 365 Lys Pro Arg Ile Glu Lys Phe Ser Leu Tyr Glu Tyr Asp Ala Leu Ser370 375 380 Phe Tyr Ser Gln Leu Gln Glu Leu Val Ser Lys Pro Asn Ser IleLys 385 390 395 400 Asp Leu Val Asn Ala Thr Leu Ala Arg Asn Leu Arg PheSer Leu Gly 405 410 415 Lys Tyr Asn Phe Leu Phe Asp Asp Leu Ala Ser HisLeu Asp Tyr Thr 420 425 430 Phe Leu Val Ser Lys Ala Lys Ile Lys Gln SerSer Ile Thr Lys Lys 435 440 445 Leu Phe Ile Glu Leu Pro Ile Lys Ile SerLeu Lys Ser Ser Ile Leu 450 455 460 Gly Asp Gln Glu Pro Asn Ile Lys ThrLeu Phe Glu Lys Glu Val Thr 465 470 475 480 Phe Lys Leu Asp Asn Phe ArgAsp Val Glu Ile Glu Lys Ala Phe Gly 485 490 495 Leu Leu Tyr Pro Gly ValAsn Glu Glu Leu Glu Gln Ala Arg Arg Glu 500 505 510 Gln Arg Ala Ser LeuGlu Lys Glu Lys Ala Lys Lys Gly Leu Lys Glu 515 520 525 Phe Ser Gln GlnLys Asp Glu Asn Leu Lys Ala Ile Asn Asn Gln Asp 530 535 540 Gly Leu GluGlu Asp Asp Asn Ile Thr Glu Arg Leu Pro Glu Asn Ser 545 550 555 560 ProIle Gln Tyr Gln Gln Glu Lys Ala Gly Leu Gly Ser Ser Pro Asp 565 570 575Lys Pro Tyr Met Ile Lys Asp Val Gln Asn Gln Arg Tyr Tyr Leu Ala 580 585590 Lys Ser Gln Ile Gln Glu Leu Ile Lys Ala Lys Asp Tyr Thr Lys Leu 595600 605 Ala Lys Leu Leu Ser Asn Arg His Thr Tyr Asn Ile Ser Leu Arg Leu610 615 620 Lys Glu Gln Leu Phe Glu Val Asn Pro Arg Ile Pro Ser Ser ArgAsp 625 630 635 640 Ile Glu Asn Ala Lys Phe Val Leu Asp Lys Thr Glu LysAsn Lys Tyr 645 650 655 Trp Gln Ile Tyr Ser Ser Ala Ser Pro Ala Phe GlnAsn Lys Trp Ser 660 665 670 Leu Phe Gly Tyr Tyr Arg Tyr Leu Leu Gly LeuAsp Pro Lys Gln Thr 675 680 685 Ile His Glu Leu Val Lys Leu Gly Gln LysAla Gly Leu Gln Phe Glu 690 695 700 Gly Tyr Glu Asn Leu Pro Ser Asp PheAsn Leu Glu Asp Leu Lys Asn 705 710 715 720 Ile Arg Ile Lys Thr Pro LeuPhe Ser Gln Lys Asp Asn Phe Lys Leu 725 730 735 Ser Leu Leu Asp Phe AsnAsn Tyr Tyr Asp Gly Glu Ile Lys Ala Pro 740 745 750 Glu Phe Gly Leu ProLeu Phe Leu Pro Lys Glu Leu Arg Lys Asn Ser 755 760 765 Ser Asn Ile GlySer Ser Gln Asn Ser Asn Ser Pro Trp Glu Gln Glu 770 775 780 Ile Ile SerGln Phe Lys Asp Gln Asn Leu Ser Asn Gln Asp Gln Leu 785 790 795 800 AlaGln Phe Ser Thr Lys Ile Trp Glu Lys Ile Ile Gly Asp Glu Asn 805 810 815Glu Phe Asp Gln Asn Asn Arg Leu Gln Tyr Lys Leu Leu Lys Asp Leu 820 825830 Gln Glu Ser Trp Ile Asn Lys Thr Arg Asp Asn Leu Tyr Trp Thr Tyr 835840 845 Leu Gly Asp Lys Leu Lys Val Lys Pro Lys Asn Asn Leu Asp Ala Lys850 855 860 Phe Arg Gln Ile Ser Asn Leu Gln Glu Leu Leu Thr Ala Phe TyrThr 865 870 875 880 Ser Ala Ala Leu Ser Asn Asn Trp Asn Tyr Tyr Gln AspSer Gly Ala 885 890 895 Lys Ser Thr Ile Ile Phe Glu Glu Ile Ala Glu LeuAsp Pro Lys Val 900 905 910 Lys Glu Lys Val Gly Ala Asp Val Tyr Gln LeuLys Phe His Tyr Ala 915 920 925 Ile Gly Phe Asp Asp Asn Ala Gly Lys PheAsn Gln Glu Val Ile Arg 930 935 940 Ser Ser Ser Arg Thr Ile Tyr Leu LysThr Ser Gly Lys Ser Lys Leu 945 950 955 960 Glu Ala Asp Thr Ile Asp GlnLeu Asn Gln Ala Val Glu Asn Ala Pro 965 970 975 Leu Gly Leu Gln Ser PheTyr Leu Asp Thr Glu Arg Phe Gly Val Phe 980 985 990 Gln Lys Leu Ala ThrSer Leu Ala Val Gln His Lys Gln Lys Glu Lys 995 1000 1005 Pro Leu ProLys Lys Leu Asn Asn Asp Gly Tyr Thr Leu Ile His Asp 1010 1015 1020 LysLeu Lys Lys Pro Val Ile Pro Gln Ile Ser Ser Ser Pro Glu Lys 1025 10301035 1040 Asp Trp Phe Glu Gly Lys Leu Asn Gln Asn Gly Gln Ser Gln AsnVal 1045 1050 1055 Asn Val Ser Thr Phe Gly Ser Ile Ile Glu Ser Pro TyrPhe Ser Thr 1060 1065 1070 Asn Phe Gln Glu Glu Ala Asp Leu Asp Gln GluGly Gln Asp Asp Ser 1075 1080 1085 Lys Gln Gly Asn Lys Ser Leu Asp AsnGln Glu Ala Gly Leu Leu Lys 1090 1095 1100 Gln Lys Leu Ala Ile Leu LeuGly Asn Gln Phe Ile Gln Tyr Tyr Gln 1105 1110 1115 1120 Gln Asn Asp LysGlu Ile Glu Phe Glu Ile Ile Asn Val Glu Lys Val 1125 1130 1135 Ser GluLeu Ser Phe Arg Val Glu Phe Lys Leu Ala Lys Thr Leu Glu 1140 1145 1150Asp Asn Gly Lys Thr Ile Arg Val Leu Ser Asp Glu Thr Met Ser Leu 11551160 1165 Ile Val Asn Thr Thr Ile Glu Lys Ala Pro Glu Met Ser Ala AlaPro 1170 1175 1180 Glu Val Phe Asp Thr Lys Trp Val Glu Gln Tyr Asp ProArg Thr Pro 1185 1190 1195 1200 Leu Ala Ala Lys Thr Lys Phe Val Leu LysPhe Lys Asp Gln Ile Pro 1205 1210 1215 Val Asp Ala Ser Gly Asn Ile SerAsp Lys Trp Leu Ala Ser Ile Pro 1220 1225 1230 Leu Val Ile His Gln GlnMet Leu Arg Leu Ser Pro Val Val Lys Thr 1235 1240 1245 Ile Arg Glu LeuGly Leu Lys Thr Glu Gln Gln Gln Gln Gln Gln Gln 1250 1255 1260 Gln GlnGln Lys Lys Ala Val Arg Lys Glu Glu Glu Leu Glu Thr Tyr 1265 1270 12751280 Asn Pro Lys Asp Glu Phe Asn Ile Leu Asn Pro Leu Thr Lys Ala His1285 1290 1295 Arg Leu Thr Leu Ser Asn Leu Val Asn Asn Asp Pro Asn TyrLys Ile 1300 1305 1310 Glu Asp Leu Lys Val Ile Lys Asn Glu Ala Gly AspHis Gln Leu Glu 1315 1320 1325 Phe Ser Leu Arg Ala Asn Asn Ile Lys ArgLeu Met Asn Thr Pro Ile 1330 1335 1340 Thr Phe Ala Asp Tyr Asn Pro PhePhe Tyr Phe Asn Glu Asp Trp Arg 1345 1350 1355 1360 Asn Ile Asp Lys TyrLeu Asn Asn Lys Gly Asn Val Ser Ser Gln Gln 1365 1370 1375 Gln Gln GlnGln Gln Gln Gln Pro Gly Gly Gly Asn Gln Gly Ser Gly 1380 1385 1390 LeuIle Gln Arg Leu Asn Lys Asn Ile Lys Pro Glu Thr Phe Thr Pro 1395 14001405 Ala Leu Ile Ala Leu Lys Arg Asp Asn Asn Thr Asn Leu Ser Asn Tyr1410 1415 1420 Ser Asp Lys Ile Ile Met Ile Lys Pro Lys Tyr Leu Val GluArg Ser 1425 1430 1435 1440 Ile Gly Val Pro Trp Ser Thr Gly Leu Asp GlyTyr Ile Gly Ser Glu 1445 1450 1455 Gln Leu Lys Gly Gly Thr Ser Ser AsnGly Gln Lys Arg Phe Lys Gln 1460 1465 1470 Asp Phe Ile Gln Ala Leu GlyLeu Lys Asn Thr Glu Tyr His Gly Lys 1475 1480 1485 Leu Gly Leu Ser IleArg Ile Phe Asp Pro Gly Asn Glu Leu Ala Lys 1490 1495 1500 Ile Lys AspAla Ser Asn Lys Lys Gly Glu Glu Lys Leu Leu Lys Ser 1505 1510 1515 1520Tyr Asp Leu Phe Lys Asn Tyr Leu Asn Glu Tyr Glu Lys Lys Ser Pro 15251530 1535 Lys Ile Ala Lys Gly Trp Thr Asn Ile His Pro Asp Gln Lys GluTyr 1540 1545 1550 Pro Asn Pro Asn Gln Lys Leu Pro Glu Asn Tyr Leu AsnLeu Val Leu 1555 1560 1565 Asn Gln Pro Trp Lys Val Thr Leu Tyr Asn SerSer Asp Phe Ile Thr 1570 1575 1580 Asn Leu Phe Val Glu Pro Glu Gly SerAsp Arg Gly Ser Gly Ala Lys 1585 1590 1595 1600 Leu Lys Gln Val Ile GlnLys Gln Val Asn Asn Asn Tyr Ala Asp Trp 1605 1610 1615 Gly Ser Ala TyrLeu Thr Phe Trp Tyr Asp Lys Asp Ile Ile Thr Asn 1620 1625 1630 Gln ProAsn Val Ile Thr Ala Asn Ile Ala Asp Val Phe Ile Lys Asp 1635 1640 1645Val Lys Glu Leu Glu Asp Asn Thr Lys Leu Ile Ala Pro Asn Ile Thr 16501655 1660 Gln Trp Trp Pro Asn Ile Ser Gly Ser Lys Glu Lys Phe Tyr LysPro 1665 1670 1675 1680 Thr Val Phe Phe Gly Asn Trp Glu Asn Glu Asn SerAsn Met Asn Ser 1685 1690 1695 Gln Gly Gln Thr Pro Thr Trp Glu Lys IleArg Glu Gly Phe Ala Leu 1700 1705 1710 Gln Ala Leu Lys Ser Ser Phe AspGln Lys Thr Arg Thr Phe Val Leu 1715 1720 1725 Thr Thr Asn Ala Pro LeuPro Leu Trp Lys Tyr Gly Pro Leu Gly Phe 1730 1735 1740 Gln Asn Gly ProAsn Phe Lys Thr Gln Asp Trp Arg Leu Val Phe Gln 1745 1750 1755 1760 AsnAsp Asp Asn Gln Ile Ala Ala Leu Arg Val Gln Glu Gln Asp Arg 1765 17701775 Pro Glu Lys Ser Ser Glu Asp Lys Asp Lys Gln Lys Trp Ile Lys Phe1780 1785 1790 Lys Val Val Ile Pro Glu Glu Met Phe Asn Ser Gly Asn IleArg Phe 1795 1800 1805 Val Gly Val Met Gln Ile Gln Gly Pro Asn Thr LeuTrp Leu Pro Val 1810 1815 1820 Ile Asn Ser Ser Val Ile Tyr Asp Phe TyrArg Gly Thr Gly Asp Ser 1825 1830 1835 1840 Asn Asp Val Ala Asn Leu AsnVal Ala Pro Trp Gln Val Lys Thr Ile 1845 1850 1855 Ala Phe Thr Asn AsnAla Phe Asn Asn Val Phe Lys Glu Phe Asn Ile 1860 1865 1870 Ser Lys LysIle Val Glu 1875 21 45 DNA Artificial Sequence Oligonucleotide 21gataatttta aaaaatggtc ggcaaaaaca gttttaactg ctgcc 45 22 45 DNAArtificial Sequence Oligonucleotide 22 ggcagcagtt aaaactgttt ttgccgaccattttttaaaa ttatc 45 23 38 DNA Artificial Sequence Oligonucleotide 23gaaagaggaa gtaattggtt ttcacgactt gaaagagc 38 24 38 DNA ArtificialSequence Oligonucleotide 24 gctctttcaa gtcgtgaaaa ccaattactt cctctttc 3825 41 DNA Artificial Sequence Oligonucleotide 25 ctaaaattct aaaatcctggcttgaaacaa atcttcaagg c 41 26 41 DNA Artificial Sequence Oligonucleotide26 gccttgaaga tttgtttcaa gccaggattt tagaatttta g 41 27 34 DNA ArtificialSequence Oligonucleotide 27 gcctctctga ttattggtat ggatctccga attc 34 2834 DNA Artificial Sequence Oligonucleotide 28 gaattcggag atccataccaataatcagag aggc 34 29 31 DNA Artificial Sequence Oligonucleotide 29gggacaagca tttggacagc ttttaatttc g 31 30 31 DNA Artificial SequenceOligonucleotide 30 cgaaattaaa agctgtccaa atgcttgtcc c 31 31 18 DNAArtificial Sequence Oligonucleotide 31 tccgacgatg acgataag 18 32 18 DNAArtificial Sequence Oligonucleotide 32 tggaaaatta gttcttgg 18 33 18 DNAArtificial Sequence Oligonucleotide 33 agtttccact tcatcgcc 18 34 15 PRTArtificial Sequence Tryptic peptide fragment 34 Glu Leu Glu Asp Asn ThrLys Leu Ile Ala Pro Asn Ile Arg Gln 1 5 10 15 35 36 DNA ArtificialSequence Oligonucleotide 35 gaantngaag ataatacnaa attaattgcn cctaat 3636 12 PRT Artificial Sequence N-terminal peptide 36 Asp Phe Leu Thr AsnAsn Gly Arg Thr Val Leu Glu 1 5 10 37 32 DNA Artificial SequenceOligonucleotide 37 gaacaatttg atcacaagat cctgaatata cc 32 38 35 DNAArtificial Sequence Oligonucleotide 38 aattcctctg atcattattt agattttaattcctg 35 39 9 PRT Artificial Sequence Antigen 39 Thr Ser Ser Gln Lys AspPro Ser Thr 1 5 40 12 PRT Artificial Sequence Antigen 40 Val Asn Gln AsnPhe Lys Val Lys Phe Gln Ala Leu 1 5 10 41 7 PRT Artificial SequenceN-terminal peptide 41 Ala Asp Glu Lys Thr Ser Ser 1 5 42 7 PRTArtificial Sequence N-terminal peptide 42 Ser Lys Lys Ser Lys Thr Phe 15

What is claimed is:
 1. A purified immunogenic polypeptide, the aminoacid sequence of which comprises at least eight consecutive residues ofa sequence selected from the group consisting of SEQ ID NOs:2, 4, 6, 8,10, 12, 14, 16, 18, and
 20. 2. The immunogenic polypeptide of claim 1,the amino acid sequence of which comprises at least 12 consecutiveresidues of a sequence selected from the group consisting of SEQ IDNOs:2, 4, 6, 8, 10, 12, 14, 16, 18, and
 20. 3. A composition comprisingthe immunogenic polypeptide of claim
 1. 4. A mutant of the immunogenicpolypeptide of claim 1, wherein said mutant polypeptide retainsimmunogenicity.
 5. A composition comprising the mutant polypeptide ofclaim
 4. 6. A method of eliciting an immune response in an animal, saidmethod comprising introducing the composition of claim 3 into saidanimal.
 7. The method of claim 6, wherein said composition isadministered orally, intranasally, intraperitoneally, intramuscularly,subcutaneously, or intravenously.
 8. The method of claim 6, wherein saidanimal is a swine.
 9. An isolated nucleic acid comprising a nucleotidesequence that encodes an immunogenic polypeptide, the amino acidsequence of which comprises at least eight consecutive residues of asequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8,10, 12, 14, 16, 18, and
 20. 10. The nucleic acid of claim 9, whereinsaid nucleotide sequence is selected from the group consisting of SEQ IDNOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, and
 19. 11. A vector comprising thenucleic acid of claim
 9. 12. A host cell comprising the vector of claim11.
 13. A mutant of the nucleic acid of claim
 9. 14. A vector comprisingthe mutant nucleic acid of claim
 13. 15. A host cell comprising thevector of claim
 14. 16. The vector of claim 11, wherein said nucleicacid is operably linked to an expression control sequence.
 17. A hostcell comprising the vector of claim
 16. 18. A composition comprising thevector of claim 16 and a pharmaceutically acceptable carrier.
 19. Amethod of eliciting an immune response in an animal, said methodcomprising introducing the composition of claim 18 into said animal. 20.A method of determining whether or not an animal has an antibodyreactive to the immunogenic polypeptide of claim 1, said methodcomprising: providing a test sample from said animal; contacting saidtest sample with said immunogenic polypeptide under conditionspermissible for specific binding of said immunogenic polypeptide withsaid antibody; and detecting the presence or absence of said specificbinding, wherein said presence of specific binding indicates that saidanimal has said antibody, and wherein said absence of specific bindingindicates that said animal does not have said antibody.
 21. The methodof claim 20, wherein said test sample is a biological fluid.
 22. Themethod of claim 21, wherein said biological fluid is selected from thegroup consisting of blood, nasal fluid, throat fluid, and lung fluid.23. The method of claim 20, wherein said immunogenic polypeptide isattached to a solid support.
 24. The method of claim 23, wherein saidsolid support is a microtiter plate, or polystyrene beads.
 25. Themethod of claim 20, wherein said immunogenic polypeptide is labeled. 26.The method of claim 20, wherein said detecting is by radioimmunoassay(RIA), enzyme immunoassay (EIA), or enzyme-linked immunosorbent assay(ELISA).
 27. A diagnostic kit for detecting the presence of an antibodyin a test sample, wherein said antibody is reactive to the immunogenicpolypeptide of claim 1, said kit comprising the immunogenic polypeptideof claim 1.